Methods and compositions for immune protection against extra-intestinal pathogenic e. coli

ABSTRACT

Compositions and methods are described for inducing an immune response against extra-intestinal pathogenic  Escherichia coli  (ExPEC) to thereby provide immune protection against diseases associated with ExPEC. In particular, compositions and methods are described for using conjugates of  E. coli  polysaccharide antigens O25B, O1A, O2, and O6A covalently bound to a detoxified exotoxin A of  Pseudomonas aeruginosa  (EPA) carrier protein as vaccines for the prevention of invasive ExPEC disease caused by ExPEC serotypes O1A, O2, O6A and O25B.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/754,867, filed Feb. 23, 2018, granted, which is a Section 371 ofInternational Application No. PCT/US2016/048278, filed Aug. 24, 2016,which was published in the English language on Mar. 2, 2017, underInternational Publication No. WO 2017/035181, which claims priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.62/209,091 filed on Aug. 24, 2015, and to U.S. Provisional PatentApplication No. 62/210,655, filed on Aug. 27, 2015, the disclosures ofall of which are herein incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “688097-86_Sequence Listing” and a creation date of Aug. 15,2016 and having a size of 6.8 KB. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods for inducing animmune response against extra-intestinal pathogenic Escherichia coli(ExPEC) to thereby provide immune protection against diseases associatedwith ExPEC. In particular, embodiments of this invention relate tomultivalent vaccines containing conjugates of E. coli polysaccharideantigens O25B, O1A, O2, and O6A each covalently bound to a detoxifiedexotoxin A of Pseudomonas aeruginosa carrier protein and uses of thevaccines to provide effective immune protection against ExPEC infection.

BACKGROUND OF THE INVENTION

Extra-intestinal pathogenic E. coli (ExPEC) are normally harmlessinhabitants of human gut. However, ExPEC strains can possess virulencefactors for the colonization and infection of sites outside of thegastrointestinal tract to cause diverse and serious invasive diseases,resulting in significant morbidity, mortality, and costs annually (see,e.g., Johnson et al., J Lab Clin Med. 2002; 139(3):155-162; Kohler etal., Int J Med Microbiol. 2011; 301(8):642-647; Foxman, Am J Med.2002;113 Suppl 1A:5S-13S; and Russo et al., Microbes Infect. 2003;5(5):449-456). ExPEC strains are the most common cause of urinary tractinfection (UTI). They are also a contributor to surgical site infectionsand neonatal meningitis (Johnson et al., 2002; and Russo et al., 2003),associated with abdominal and pelvic infections and nosocomialpneumonia, and are occasionally involved in other extra-intestinalinfections such as osteomyelitis, cellulitis, and wound infections. Allthese primary sites of infection can result in ExPEC bacteremia (Russoet al., 2003).

Bacterial resistance to antibiotics is a major concern in the fightagainst bacterial infection, and multi-drug resistant (MDR) E. colistrains are becoming more and more prevalent (see, e.g., Schito et al.,2009, Int. J. Antimicrob. Agents 34(5):407-413; and Pitout et al., 2012,Expert Rev. Anti. Infect. Ther. 10(10):1165-1176). The emergence andrapid global dissemination of ExPEC sequence type 131 is considered themain driver of increased drug resistance, including multi-drugresistance (Johnson et al., Antimicrob Agents Chemother. 2010;54(1):546-550; Rogers et al., J Antimicrob Chemother. 2011; 66(1):1-14).This clone is found in 12.5% to 30% of all ExPEC clinical isolates,mostly exhibits serotype O25B:H4, and shows high levels offluoroquinolone resistance, which is often accompanied bytrimethoprim/sulfamethoxazole resistance (Rogers et al, 2011, andBanerjee et al., Antimicrob Agents Chemother. 2014; 58(9):4997-5004).

The O-antigen serotype is based on the chemical structure of the Opolysaccharide antigen, the outer membrane portion of thelipopolysaccharide (LPS) in a Gram-negative bacterium. More than 180 E.coli O-antigens have been reported (Stenutz et al., FEMS Microbial Rev.2006; 30: 382-403). ExPEC infection can be caused by any serotype.Although there is an overrepresentation of certain serotypes in ExPECinfection, surface polysaccharides from ExPEC isolates nonethelessexhibit considerable antigenic diversity, which makes the development ofan ExPEC vaccine based on surface polysaccharides extremely challenging(Russo et al., Vaccine. 2007; 25: 3859-3870). Also, certain O-antigensmay be poorly immunogenic. Furthermore, based on studies fromPneumococcal conjugate vaccines, when a number of serotypes can cause adisease, the vaccine composition, such as the choice of serotypes forinclusion in a vaccine and the dosage levels of the included serotypes,can be critical, since use of a vaccine against certain serotypes maypotentially increase carriage of and disease from serotypes not includedin the vaccine, or even a serotype that is included in the vaccine butonly weakly effective in immunizing against the serotype (Lipsitch,Emerging Infectious Diseases; 1999, 5:336-345). Ideally, a vaccineshould maximize its beneficial effects in the prevention of diseasecaused by serotypes included in the vaccine, while minimizing the riskof added disease from increased carriage of non-vaccine serotypes.

Accordingly, there is a need in the art for vaccines against ExPEC. Inparticular, there exists a need for an ExPEC vaccine based on surfacepolysaccharides that can be used to provide effective immune protectionagainst ExPEC O25B serotype and other serotypes prevalent among ExPEC.

BRIEF SUMMARY OF THE INVENTION

It has been surprisingly discovered that an E. coli O25B antigen appearsto be somewhat less immunogenic than other E. coli O-antigens (e.g.,O1A, O2, and O6A) when tested as conjugates of the O-antigens eachcovalently bound to a detoxified exotoxin A of P. aeruginosa (EPA)carrier protein, and that vaccination with a composition containing EPAconjugates of E. coli O25B antigen and EPA conjugates of one or moreadditional E. coli O-antigens at an appropriate dose and ratio providesan improved immune response against the ExPEC O25B serotype and the oneor more additional ExPEC O-serotypes.

Accordingly, in one general aspect, the invention relates to acomposition comprising an E. coli O25B antigen at a first concentrationof 8 to 48 μg/ml, and at least one additional E. coli O-antigen at asecond concentration that is 10% to 100% of the first concentration,wherein each of the E. coli O25B antigen and the at least one additionalE. coli O-antigen is independently covalently bound to an EPA carrierprotein, and the composition is effective in inducing an immune responseagainst the E. coli O25B antigen and the at least one additional E. coliO-antigen in a subject in need thereof. In a preferred embodiment, theat least one additional E. coli O-antigen is present at a secondconcentration of at least 5 μg/ml, more preferably, at a secondconcentration of least 8 μg/ml.

In one embodiment, the invention relates to a composition comprising anE. coli O25B antigen at a first concentration of 10 to 36 μg/ml, and atleast one additional E. coli O-antigen selected from the groupconsisting of an E. coli O1A antigen, an E. coli O2 antigen and an E.coli O6A antigen, wherein each of the additional E. coli O-antigens hasa concentration that is independently 50% to 100% of the firstconcentration of the E. coli O25B antigen in the composition, and eachof the E. coli O25B, O1A, O2 and O6A antigens are independentlycovalently bound to an EPA carrier protein.

In one preferred embodiment, the invention relates to a compositioncomprising an E. coli O25B antigen at a first concentration of 10 to 36μg/ml, and a second concentration of each of an E. coli O1A antigen, anE. coli O2 antigen and an E. coli O6A antigen, wherein each of the E.coli O25B, O1A, O2 and O6A antigens are independently covalently boundto an EPA carrier protein, and the ratio of the first concentration tothe second concentration is 1:1 to 2:1. Preferably, the compositioncomprises the E. coli O25B, O1A, O2 and O6A antigen polysaccharides at aweight ratio of 1:1:1:1 or 2:1:1:1. More preferably, the compositioncomprises 16 or 32 μg/ml of the O25B antigen.

In another general aspect, the invention relates to a method of inducingan immune response to extra-intestinal pathogenic E. coli (ExPEC) in asubject in need thereof. The method comprises administering to thesubject an E. coli O25B antigen at a first effective amount of 4 to 24μg per administration, and at least one additional E. coli O-antigen ata second effective amount that is 10% to 100% of the first effectiveamount, wherein each of the E. coli O25B antigen and the at least oneadditional E. coli O-antigen is independently covalently bound to an EPAcarrier protein, and the administration is effective in inducing animmune response against the E. coli O25B antigen and the at least oneadditional E. coli O-antigen in the subject. In a preferred embodiment,the at least one additional E. coli O-antigen is administered at asecond effective amount of at least 3 μg per administration, morepreferably, at a second effective amount of least 4 μg peradministration. The E. coli O25B antigen and the at least one additionalE. coli O-antigen can be administered in one composition or administeredin combination from multiple compositions.

In one embodiment, the invention relates to a method of inducing animmune response to ExPEC in a subject in need thereof, comprisingadministering to the subject an E. coli O25B antigen at a firsteffective amount of 5 to 18 μg per administration, more preferably 8 to16 μg per administration, and at least one additional E. coli O-antigenselected from the group consisting of an E. coli O1A antigen, an E. coliO2 antigen and an E. coli O6A antigen, wherein each of the additional E.coli O-antigens is administered at an effective amount that isindependently 50% to 100% of the first effective amount of the E. coliO25B antigen, and each of the E. coli O25B, O1A, O2 and O6A antigens areindependently covalently bound to an EPA carrier protein.

In one preferred embodiment, the invention relates to a method ofinducing an immune response to ExPEC in a subject in need thereof,comprising administering to the subject an E. coli O25B antigen at afirst effective amount of 5 to 18 μg per administration, more preferably8 to 16 μg per administration, and a second effective amount of each ofan E. coli O1A antigen, an E. coli O2 antigen and an E. coli O6Aantigen, wherein each of the E. coli O25B, O1A, O2 and O6A antigens areindependently covalently bound to an EPA carrier protein, and the ratioof the first effective amount to the second effective amount is 1:1 to2:1. Preferably, the E. coli O25B, O1A, O2 and O6A antigens areadministered at a dosage ratio of 1:1:1:1 or 2:1:1:1, and the E. coliO25B antigen is administered at 5 μg, 8 μg or 16 μg per administration.In a preferred embodiment, the E. coli O25B, O1A, O2 and O6A antigensare administered to the subject in one composition, more preferably, incompositions comprising the E. coli O25B, O1A, O2 and O6A antigens eachindependently covalently bound to an EPA carrier protein, wherein theconcentrations of these O-antigens in the compositions are chosen fromrespectively 16:8:8:8 μg/ml (i.e., 16 μg/ml of O25B antigen, 8 μg/ml ofO1A antigen, 8 μg/ml of O2 antigen, and 8 μg/ml of O6A antigen),16:16:16:16 μg/ml, 32:16:16:16 μg/ml and 32:32:32:32 μg/ml. Preferably,0.5 ml of such a composition is administered to a subject to achieve adosage per administration of E. coli O25B, O1A, O2 and O6A antigensrespectively at 8:4:4:4 μg (i.e., 8 μg of O25B antigen, 4 μg of O1Aantigen, 4 μg of O2 antigen, and 4 μg of O6A antigen), 8:8:8:8 μg,16:8:8:8 μg or 16:16:16:16 μg.

According to a preferred embodiment, the immune response induced by amethod of the present invention prevents an invasive ExPEC diseasecaused by ExPEC serotypes O1A, O2, O6A and O25B in a human subject inneed thereof. Diseases associated with ExPEC or ExPEC diseases include,but are not limited to, urinary tract infection, surgical-siteinfection, bacteremia, abdominal or pelvic infection, pneumonia,nosocomial pneumonia, osteomyelitis, cellulitis, pyelonephritis, woundinfection, meningitis, neonatal meningitis, peritonitis, cholangitis,soft-tissue infections, pyomyositis, septic arthritis, and sepsis.Preferably, the human subject is an at-risk human subject, who has or isat risk of having an invasive ExPEC disease, such as an elderly human, ahospitalized patient, a human child, an immunocompromised human, apregnant woman, people with diabetes or wound injuries, people whorecently had or are scheduled to have a surgery, etc.

According to an embodiment of the invention, each of the O-antigen iscovalently bound to the EPA carrier protein at the Asn residue of aglycosylation sequence comprising Asp (Glu)-X-Asn-Z-Ser (Thr) (SEQ IDNO: 3), wherein X and Z are independently selected from any naturalamino acid except Pro. In a preferred embodiment, the EPA carrierprotein comprises the amino acid sequence of SEQ ID NO: 1. In anotherembodiment, the O-antigen is covalently bound to the EPA carrier proteinat a polysaccharide-to-protein weight ratio of 1:7 to 1:2, preferably,1:5 to 1:2. For example, in an O-antigen/EPA conjugate according to anembodiment of the invention, the weight of the O-antigen can be 15% to50%, or 20% to 40%, of the weight of the EPA.

Another aspect of the invention relates to a process of making acomposition according to an embodiment of the invention, the processcomprises combining the E. coli O25B, O1A, O2 and O6A antigens, eachindependently covalently bound to the EPA carrier protein, in onecomposition.

Yet another aspect of the invention relates to use of a compositionaccording to an embodiment of the invention for the manufacture of avaccine or medicament for inducing an immune response to ExPEC or forpreventing or treating a disease associated with ExPEC in a subject inneed thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood that the invention is notlimited to the precise embodiments shown in the drawings.

In the drawings:

FIG. 1 is an exemplary representation of the glycoconjugate vaccineproduction platform: the cytoplasm (marked in grey) of the host cellcontains all the DNA constructs necessary for the recombinant productionof the O-antigen/EPA conjugate in the periplasm (marked in white) of thehost cell;

FIG. 2 is a detailed schematic representation of the proteinglycosylation process;

FIGS. 3A-3C depict the opsonization indices (OIs) obtained with seraderived from rats pre-immunization (empty circles) compared to 42 dayspost-immunization (filled squares) with one priming dose and two boosterdoses of indicated doses of monovalent vaccine; FIG. 3A: O2-EPAimmunization; FIG. 3B: O6A-EPA immunization; and FIG. 3C: O25B-EPAimmunization;

FIG. 4 shows the ELISA titers obtained with sera from human subjectsvaccinated with a placebo or a tetravalent vaccine comprising E. coliantigens O1A, O2, O6A and O25B at 4 μg polysaccharide per serotype; asignificant increase in the ELISA titers between post- (30 days afterinjection) and pre-injection (day 1) was observed only in the vaccinatedgroups (* represents statistical significance, wherein multiple *represent increased degree of significance; ns, no significantdifference); and

FIGS. 5A-5D depict the OIs obtained with sera derived from humansubjects vaccinated with a tetravalent vaccine comprising E. coliantigens O1A, O2, O6A and O25B at 4 μg polysaccharide per serotype;immune response as indicated by OI against placebo and components of thetetravalent vaccine; FIG. 5A: O1A-EPA; FIG. 5B: O2-EPA; FIG. 5C:O6A-EPA; and FIG. 5D: O25B-EPA; pre-injection (Day 1) and post-injection(30 days after injection), wherein a significant increase in the OIbetween post- and pre-injection (indicated by *, multiple * representsincreased degree of significance) was observed only in the vaccinatedgroups; ns, no significant difference.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms citedherein have the meanings as set in the specification. All patents,published patent applications and publications cited herein areincorporated by reference as if set forth fully herein. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

As used herein, the terms “O polysaccharide,” “O-antigen”, “O antigen”,“O-antigen polysaccharide,” “O-polysaccharide antigen” and theabbreviation “OPS”, all refer to the O antigen of Gram-negativebacteria, which is a component of the lipopolysaccharide (LPS) and isspecific for each serotype or sero(sub)type of the Gram-negativebacteria. The O antigen usually contains repeating units (RUs) of two toseven sugar residues. As used herein, the RU is set equal to thebiological repeat unit (BRU). The BRU describes the RU of an O-antigenas it is synthesized in vivo.

As used herein, the terms “conjugate” and “glycoconjugate” all refer toa conjugation product containing an E. coli O antigen covalently boundto a carrier protein. The conjugate can be a bioconjugate, which is aconjugation product prepared in a host cell, wherein the host cellmachinery produces the O antigen and the carrier protein and links the Oantigen to the carrier protein, e.g., via N-links. The conjugate canalso be prepared by other means, for example, by chemical linkage of theprotein and sugar antigen.

As used herein, the term “effective amount” in the context ofadministering an O antigen to a subject in methods according toembodiments of the invention refers to the amount of the O antigen thatis sufficient to induce a desired immune effect or immune response inthe subject. In certain embodiments, an “effective amount” refers to theamount of an O antigen which is sufficient to produce immunity in asubject to achieve one or more of the following effects in the subject:(i) prevent the development or onset of an ExPEC infection, preferablyan invasive ExPEC disease, or symptom associated therewith; (ii) preventthe recurrence of an ExPEC infection, preferably an invasive ExPECdisease, or symptom associated therewith; (iii) prevent, reduce orameliorate the severity of an ExPEC infection, preferably an invasiveExPEC disease, or symptom associated therewith; (iv) reduce the durationof an ExPEC infection, preferably an invasive ExPEC disease, or symptomassociated therewith; (v) prevent the progression of an ExPEC infection,preferably an invasive ExPEC disease, or symptom associated therewith;(vi) cause regression of an ExPEC infection or symptom associatedtherewith; (vii) prevent or reduce organ failure associated with anExPEC infection; (viii) reduce the chance or frequency ofhospitalization of a subject having an ExPEC infection; (ix) reducehospitalization length of a subject having an ExPEC infection; (x)increase the survival of a subject with an ExPEC infection, preferablyan invasive ExPEC disease; (xi) eliminate an ExPEC infection, preferablyan invasive ExPEC disease; (xii) inhibit or reduce ExPEC replication;and/or (xiii) enhance or improve the prophylactic or therapeuticeffect(s) of another therapy.

An “effective amount” can vary depending upon a variety of factors, suchas the physical condition of the subject, age, weight, health, etc.;route of administration, such as oral or parenteral; the compositionadministered, such as the target O antigen, the other co-administered Oantigens, adjuvant, etc.; and the particular disease for which immunityis desired. When the O antigen is covalently bound to a protein carrier,the effective amount for the O antigen is calculated based on only the Oantigen polysaccharide moiety in the conjugate.

As used herein, the term “in combination,” in the context of theadministration of two or more O antigens or compositions to a subject,does not restrict the order in which O antigens or compositions areadministered to a subject. For example, a first composition can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, orsubsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours,72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks,8 weeks, or 12 weeks after) the administration of a second compositionto a subject.

As used herein, “subject” means any animal, preferably a mammal, mostpreferably a human, to who will be or has been vaccinated by a method orcomposition according to an embodiment of the invention. The term“mammal” as used herein, encompasses any mammal. Examples of mammalsinclude, but are not limited to, cows, horses, sheep, pigs, cats, dogs,mice, rats, rabbits, guinea pigs, monkeys, humans, etc., most preferablya human. In some embodiments, a subject is a human infant. In anotherembodiment, a subject is a human child. In another embodiment, a subjectis a human adult. In a specific embodiment, a subject is an at-riskhuman adult. In another embodiment, a subject is an elderly human. Inanother embodiment, a subject is a human infant, including a prematurehuman infant and a human infant born at term. In another embodiment, asubject is a human toddler. The terms “subject” and “patient” may beused herein interchangeably.

As used herein, the term “premature human infant” refers to a humaninfant born at less than 37 weeks of gestational age.

As used herein, the term “human infant” refers to a newborn to 1 yearold human.

As used herein, the term “human toddler” refers to a human that is 1year to 3 years old.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “at-risk human adult” refers to a human that is18 years or older who is more prone to ExPEC infection than the averagehuman adult population. Examples of “at-risk human adult” include, butnot limited to, elderly humans, immunocompromised humans, pregnantwomen, people with diabetes or wound injuries, people who recently hador are scheduled to have a surgery, etc.

As used herein, the term “elderly human” refers to a human that is 55,preferably 60, more preferably 65, years or older.

As used herein, an “invasive ExPEC disease” is defined as isolation andidentification of ExPEC from normally sterile body sites in a subjectpresenting with an acute illness consistent with bacterial infection.

As used herein, an “immunological response” or “immune response” to anantigen or composition refers to the development in a subject of ahumoral and/or a cellular immune response to the antigen or an antigenpresent in the composition.

It has been surprisingly discovered in the invention that E. coli O25Bantigen conjugated to an EPA carrier protein appears to be lessimmunogenic than the other E. coli O-antigens (e.g., O1A, O2, and O6A)conjugated to the EPA carrier protein. This discovery leads to furtherinvestigation into the dosage of E. coli O25B antigen and the dosageratios of various E. coli O antigens within a multivalent vaccine, thusthe development of multivalent vaccines and immunization methods basedon E. coli O antigens for improved immune responses against the O25Bserotype and other serotypes of ExPEC.

Epidemiology

Studies on the serotype distribution of E. coli causing ExPEC diseaseindicate that 10 predominant O serotypes could cover an estimated 60-80%of ExPEC infections, assuming coverage of subportions of thenon-typeable strains. See, e.g., Tables 1A-1C below. In both UTI andbacteremia target populations, serotypes O1, O2, O6, and O25 wereidentified as the four most prevalent E. coli serotypes, among which,serotype O25 was the most prevalent E. coli serotype in the bacteremia.It was also found that, for an O antigen serotype that is composed ofdistinct, yet structurally and antigenically related subtypes, onesubtype may be more prevalent among the clinical isolates than theothers. For example, O1A, O6A and O25B antigens were determined to bethe more frequent subtypes among the analyzed more recent clinicalstrains or isolates for O1, O6 and O25 serotypes, respectively. Seerelated disclosure on epidemiology studies in International Patentapplication No. PCT/EP2015/053739, the disclosure of which is hereinincorporated by reference in its entirety.

TABLE 1A Distribution of the most common UTI-associated E. coliserotypes from a collection of 1841 urine samples collected inSwitzerland in 2012. Shown is the serotype distribution of samples froma relevant sub- population of 671 subjects, and the distribution fromall** samples. Most prevalent E. coli serotypes associated with UTICommunity acquired Community and hospital O- UTI in 18-70 years old* O-acquired UTI in serotype (n = 671) serotype all ages ** (n = 1871) 610.75% 2 8.75% 2 9.55% 6 8.47% 25 6.87% 25 8.37% 1 5.52% 75 4.56% 45.37% 1 4.29% 75 4.78% 8 3.86% 8 3.43% 18 3.53% 18 3.28% 4 3.26% 153.28% 15 2.39% 73 2.24% 73 2.17% 16 2.24% 16 1.85% 7 1.94% 7 1.68%

TABLE 1B Prevalence of most common UTI-associated serotypes fromselected literature ranging from 1987-2011 and from retrospectivelyanalyzed US data from 2000-2011 (ECRC). US TOTAL PYELONE- 2000-2010 UTICYSTITIS PHRITIS 315 (all UTI available available available specimendata from data from data from except INDI- 1860 1089 373 fecal, allCATION isolates isolates isolates ages, F + M)* Serotype O1 4.8% 4.1%5.4% 7.0% O2 7.1% 4.9% 15.3% 14.0% O4 7.8% 6.0% 3.2% 3.2% O6 16.9% 16.3%7.8% 18.7% O7 3.3% 2.4% 2.4% 1.9% O8 1.7% 3.2% 0.8% 3.5% O15 0.6% 1.5%0.8% 1.3% O16 4.3% 3.2% 7.2% 1.9% O18 7.0% 7.1% 6.7% 7.0% O21 Na Na Na1.3% O22 0.6% 0.6% 0.5% 0.0% O25 3.0% 4.8% 0.5% 8.6% O75 7.5% 6.0% 8.6%3.8% O83 1.9% 0.7% 0.5% 1.3% O20 1.6% O77 2.2% O82 1.9% others 33.3%39.2% 40.2% and non typeable/not available other O-types 21.0% (NT notavailable) *Number of non-typeable was not available

TABLE 1C Distribution of the most common bacteremia-associated E. coliO-serotypes from a collection of 860 blood isolates collected in the USand EU in the period 2011-2013. Indicated is the relative O-serotypedistribution of the samples. O- Bacteremia in ≥ 60 years old US/EUserotype 2011-2013 (n = 860) 25 19.2 2 8.8 6 8.3 1 7.8 75 3.3 4 2.8 162.7 18 2.7 15 2.3 8 2.0 153 1.6 73 1.6

A novel O25 agglutinating clone has recently emerged in E. coli isolatedfrom hospital settings, and this is named O25B. For O-serotype O25, itwas found using subtyping analysis by PCR that the vast majority isactually of the O25B subtype (in a study of 24 tested clinical isolateswith an O25 agglutination positive phenotype, 20 were assigned to theO25B subtype while the remaining 4 were assigned to the O25A subtype,and in the bacteremia population that was studied then, 56 of 57 studiedO25 serotype isolates were typeable as O25B). It has been confirmed thatautologous antisera recognize the autologous antigen better than thenon-autologous antigen, and therefore inclusion of the O25B antigen intoa vaccine can provide better protection against the predominant O25Bclinical strains of the O25 group than inclusion of the O25A antigenwould do (see, e.g., International Patent application No.PCT/EP2015/053739). Results showed that an O25B vaccine can give rise tosera that cross-react with an O25A antigen, thus also providing immuneresponse against O25A serotype (Id.). Compositions according toembodiments of the invention comprise an E. coli O25B antigen conjugatedto an EPA carrier protein and other E. coli O-antigens conjugated to theEPA carrier protein.

Compositions Comprising E. coli O Antigen Conjugates

In one general aspect, the invention relates to a multivalent vaccinecontaining O-antigen serotypes found predominantly among E. coliclinical isolates, which can be used to provide active immunization forthe prevention of disease caused by ExPEC having the O-antigen serotypescontained in the vaccine. In one embodiment, the invention relates to acomposition comprising an E. coli O25B antigen at a first concentrationof 8 to 48 μg/ml, and at least one additional E. coli O-antigen at asecond concentration that is 10% to 100% of the first concentration,wherein each of the E. coli O25B antigen and the at least one additionalE. coli O-antigen is independently covalently bound to an EPA carrierprotein.

Preferably, the at least one additional E. coli O antigen used incompositions according to embodiments of the invention is prevalentamong the E. coli clinical isolates. Examples of such additional Oantigens include, but are not limited to, E. coli O1, O2, O4, O6, O7,O8, O15, O16, O18, O21, O73, O75 and O153 antigens. Depending on theneed, the composition can include more than one additional E. coli Oantigens, such as two, three, four, five, six, seven, eight or nineadditional E. coli O antigens, to provide immune protection againstmultiple E. coli serotypes in addition to E. coli O25B serotype. In apreferred embodiment, the additional E. coli O-antigen is selected fromthe group consisting of E. coli O1, O2 and O6 antigens. More preferably,the additional E. coli O-antigen is selected from the group consistingof E. coli O1A, O2 and O6A antigens.

In one embodiment, a composition of the invention comprises an E. coliO25B antigen at a first concentration of 10 to 36 μg/ml, and E. coliO1A, O2 and O6A antigens each at a concentration that is independently10% to 100% of the first concentration, wherein each of the E. coli O25Bantigen and the additional E. coli O-antigens is independentlycovalently bound to an EPA carrier protein. In a preferred embodiment,each of the E. coli O1A, O2 and O6A antigens is independently present ata concentration of at least 5 μg/ml, more preferably, at a concentrationof least 8 μg/ml.

A composition according to an embodiment of the invention contains E.coli O25B antigen at a concentration that is same or higher than theconcentration of any of the additional O antigens in the composition.For example, the composition can have E. coli O25B antigen at a firstconcentration of, e.g., 10, 16, 24, 32 or 36 μg/ml, and one or moreadditional E. coli O-antigens each at a concentration that is, e.g. 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the firstconcentration. When the composition contains more than one additional Oantigen, all of the additional O antigens can have the sameconcentration that is 10 to 100% of the E. coli O25B antigenconcentration in the composition. Alternatively, the additional Oantigens can also have different concentrations, each of which isindependently 10-100% of the E. coli O25B antigen concentration.Preferably, the composition comprises 32 μg/ml E. coli O25B antigen andindependently 16 to 32 μg/ml each of the one or more additional Oantigens, wherein each of the E. coli O25B antigen and the additional E.coli O-antigens is independently covalently bound to an EPA carrierprotein. In another preferred embodiment, the composition comprises 16μg/ml E. coli O25B antigen, and independently 8 μg/ml to 16 μg/ml ofeach of the one or more additional O antigens, wherein each of the E.coli O25B antigen and the additional E. coli O-antigens is independentlycovalently bound to an EPA carrier protein.

In one preferred embodiment, the invention relates to a compositioncomprising an E. coli O25B antigen at a first concentration of 10 to 36μg/ml, and a second concentration of each of an E. coli O1A antigen, anE. coli O2 antigen and an E. coli O6A antigen, wherein each of the E.coli O25B, O1A, O2 and O6A antigens are independently covalently boundto an EPA carrier protein, and the ratio of the first concentration tothe second concentration is 1:1 to 2:1.

Preferably, the composition comprises the E. coli O25B, O1A, O2 and O6Aantigens at a weight ratio of 1:1:1:1 or 2:1:1:1, and the compositioncomprises 32 μg/ml of the E. coli O25B antigen, wherein each of theO-antigen is covalently bound to an EPA carrier protein. In anotherpreferred embodiment, the composition comprises the E. coli O25B, O1A,O2 and O6A antigens at a weight ratio of 1:1:1:1 or 2:1:1:1, and thecomposition comprises 16 μg/ml of the E. coli O25B antigen, wherein eachof the O-antigen is covalently bound to an EPA carrier protein. Morepreferably, each of the E. coli O25B, O1A, O2 and O6A antigens areindependently covalently bound to an EPA carrier protein having theamino acid sequence of SEQ ID NO: 1, and each of the O-antigen and EPAcarrier protein conjugate is made in a cell, i.e., being a bioconjugate.Most preferably, the E. coli O25B, O1A, O2 and O6A antigens comprise,respectively, the structures of formula O25B′, formula O1A′, formula O2′and formula O6A′ described infra.

The compositions described herein are useful in the treatment andprevention of infection of subjects (e.g., human subjects) with ExPEC.In certain embodiments, in addition to comprising E. coli O-antigenscovalently bound to an EPA carrier protein, the compositions describedherein comprise a pharmaceutically acceptable carrier. As used herein,the term “pharmaceutically acceptable” means approved by a regulatoryagency of a Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier,” as usedherein in the context of a pharmaceutically acceptable carrier, refersto a diluent, adjuvant, excipient, or vehicle with which thepharmaceutical composition is administered. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Examples of suitable pharmaceutical carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin.

In a specific embodiment, provided herein is a composition comprising anE. coli O25B antigen covalently bound to an EPA carrier protein, and oneor more additional E. coli O antigens each covalently bound to the EPAcarrier protein.

In another specific embodiment, provided herein is a compositioncomprising (i) a bioconjugate comprising an E. coli O25B antigencovalently bound to an EPA carrier protein, and (ii) a bioconjugatecomprising an E. coli O1A antigen covalently bound to an EPA carrierprotein.

In another specific embodiment, provided herein is a compositioncomprising (i) a bioconjugate comprising an E. coli O25B antigencovalently bound to an EPA carrier protein, and (ii) a bioconjugatecomprising an E. coli O2 antigen covalently bound to an EPA carrierprotein.

In another specific embodiment, provided herein is a compositioncomprising (i) a bioconjugate comprising an E. coli O25B antigencovalently bound to an EPA carrier protein, and (ii) a bioconjugatecomprising an E. coli O6A antigen covalently bound to an EPA carrierprotein.

In another specific embodiment, provided herein is a compositioncomprising an E. coli O25B bioconjugate comprising an E. coli O25Bantigen covalently bound to an EPA carrier protein, and two or morebioconjugates selected from the group consisting of: (i) an E. coli O1Abioconjugate comprising an E. coli O1A antigen covalently bound to anEPA carrier protein; (ii) an E. coli O2 bioconjugate comprising an E.coli O2 antigen covalently bound to an EPA carrier protein; and (iii) anE. coli O6A bioconjugate comprising an E. coli O6A antigen covalentlybound to an EPA carrier protein.

In another specific embodiment, a composition provided herein comprises:(i) an E. coli O25B bioconjugate comprising an E. coli O25B antigencovalently bound to an EPA carrier protein; (ii) an E. coli O1Abioconjugate comprising an E. coli O1A antigen covalently bound to anEPA carrier protein; (iii) an E. coli O2 bioconjugate comprising an E.coli O2 antigen covalently bound to an EPA carrier protein; and (iv) anE. coli O6A bioconjugate comprising an E. coli O6A antigen covalentlybound to an EPA carrier protein, wherein (i), (ii), (iii), and (iv) areformulated in a single formulation.

In another specific embodiment, a composition provided herein comprises:(i) an E. coli O25B bioconjugate comprising an E. coli O25B antigencovalently bound to an EPA carrier protein; (ii) an E. coli O1Abioconjugate comprising an E. coli O1A antigen covalently bound to anEPA carrier protein; (iii) an E. coli O2 bioconjugate comprising an E.coli O2 antigen covalently bound to an EPA carrier protein; and (iv) anE. coli O6A bioconjugate comprising an E. coli O6A antigen covalentlybound to an EPA carrier protein, wherein (i), (ii), (iii), and (iv) areformulated in individual compositions that are administered incombination according to a method of an embodiment of the invention.

In certain embodiments, the foregoing compositions optionally comprisean EPA carrier protein covalently linked to an E. coli O-antigen otherthan E. coli O1A, O2, O6A, and O25B. Other E. coli O antigens include,but are not limited to, the additional O antigens listed in Tables 1A-1Cabove.

The compositions provided herein can be used for eliciting an immuneresponse in a host to whom the composition is administered, i.e., areimmunogenic. Thus, the compositions described herein can be used asvaccines against ExPEC infection, and can comprise any additionalcomponents suitable for use in a vaccine. In a specific embodiment, thecompositions described herein are multivalent formulations, e.g., atleast tetravalent formulations comprising bioconjugates of E. coliO-antigens of the O25B, O1A, O6A, and O2 serotypes/subserotypes.

In certain embodiments, the compositions described herein additionallycomprise a preservative, such as the mercury derivative thimerosal. In aspecific embodiment, the pharmaceutical compositions described hereincomprise 0.001% to 0.01% thimerosal. In other embodiments, thepharmaceutical compositions described herein do not comprise apreservative.

In certain embodiments, the compositions described herein (e.g., theimmunogenic compositions) comprise, or are administered in combinationwith, an adjuvant. The adjuvant for administration in combination with acomposition described herein may be administered before, concomitantlywith, or after administration of said composition. In some embodiments,the term “adjuvant” refers to a compound that when administered inconjunction with or as part of a composition described herein augments,enhances and/or boosts the immune response to a bioconjugate, but whenthe adjuvant compound is administered alone does not generate an immuneresponse to the bioconjugate. In some embodiments, the adjuvantgenerates an immune response to the poly bioconjugate peptide and doesnot produce an allergy or other adverse reaction. Adjuvants can enhancean immune response by several mechanisms including, e.g., lymphocyterecruitment, stimulation of B and/or T cells, and stimulation ofmacrophages. In certain preferred embodiments, the compositionsdescribed herein do not comprise an adjuvant besides the bioconjugates,and/or are not administered in combination with an adjuvant besides thebioconjugates (in case the bioconjugates would comprise some intrinsicadjuvant properties, these would be disregarded and no extrinsicadjuvant would be added in these embodiments).

Specific examples of adjuvants include, but are not limited to, aluminumsalts (alum) (such as aluminum hydroxide, aluminum phosphate, andaluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (seeUnited Kingdom Patent GB2220211), MF59 (Novartis), AS03(GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICLAmericas, Inc.), imidazopyridine compounds (see InternationalApplication No. PCT/US2007/064857, published as InternationalPublication No. WO2007/109812), imidazoquinoxaline compounds (seeInternational Application No. PCT/US2007/064858, published asInternational Publication No. WO2007/109813) and saponins, such as QS21(see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach(eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No.5,057,540). In some embodiments, the adjuvant is Freund's adjuvant(complete or incomplete). Other adjuvants are oil in water emulsions(such as squalene or peanut oil), optionally in combination with immunestimulants, such as monophosphoryl lipid A (see Stoute et al., 1997, N.Engl. J. Med. 336, 86-91). Another adjuvant is CpG (Bioworld Today, Nov.15, 1998).

In certain embodiments, the compositions described herein are formulatedto be suitable for the intended route of administration to a subject.For example, the compositions described herein may be formulated to besuitable for subcutaneous, parenteral, oral, intradermal, transdermal,colorectal, intraperitoneal, and rectal administration. In a specificembodiment, the pharmaceutical composition may be formulated forintravenous, oral, intraperitoneal, intranasal, intratracheal,subcutaneous, intramuscular, topical, intradermal, transdermal orpulmonary administration. In certain embodiments, the compositionsdescribed herein are administered by intramuscular injection.

In certain embodiments, the compositions described herein additionallycomprise one or more buffers, e.g., Tris-buffered saline, phosphatebuffer, and sucrose phosphate glutamate buffer.

In certain embodiments, the compositions described herein additionallycomprise one or more salts, e.g., Tris-hydrochloride, sodium chloride,calcium chloride, potassium chloride, sodium phosphate, monosodiumglutamate, and aluminum salts (e.g., aluminum hydroxide, aluminumphosphate, alum (potassium aluminum sulfate), or a mixture of suchaluminum salts). In one embodiment, a composition of the inventioncomprises the bioconjugates described herein in a Tris-buffered saline(TBS) pH 7.4 (e.g. containing Tris, NaCl and KCl, e.g. at 25 mM, 137 mMand 2.7 mM, respectively).

The compositions described herein can be included in a container, pack,or dispenser together with instructions for administration.

The compositions described herein can be stored before use, e.g., thecompositions can be stored frozen (e.g., at about −20° C. or at about−70° C.); stored in refrigerated conditions (e.g., at about 4° C.); orstored at room temperature.

Methods/Uses

In another general aspect, the invention relates to a method of inducingan immune response to ExPEC in a subject in need thereof. Preferably,the immune response is effective to prevent or treat a diseaseassociated with ExPEC in the subject in need thereof. The methodcomprises administering to the subject an E. coli O25B antigen at afirst effective amount of 4 to 24 μg per administration, and at leastone additional E. coli O antigen at a second effective amount that is10% to 100% of the first effective amount, wherein each of the E. coliO25B antigen and the at least one additional E. coli O-antigen isindependently covalently bound to an EPA carrier protein, and thecomposition is effective in inducing an immune response against the E.coli O25B antigen and the at least one additional E. coli O-antigen inthe subject in need thereof.

Preferably, the at least one additional E. coli O antigen used in themethods and uses of the invention is prevalent among the E. coliclinical isolates. Examples of such additional O antigens include, butare not limited to, E. coli O1, O2, O4, O6, O7, O8, O15, O16, O18, O21,O73, O75 and O153 antigens. Depending on the need, more than oneadditional E. coli O antigens, such as two, three, four, five, six,seven, eight or nine additional E. coli O antigens, can be administeredto provide immune protection against multiple E. coli serotypes inaddition to E. coli O25B serotype.

In a preferred embodiment, a method of the invention induces an immuneresponse in a subject in need thereof against ExPEC serotype O25, andone or more additional E. coli O-antigens selected from the groupconsisting of E. coli O1, O2 and O6 antigens. Preferably, a method ofthe invention induces an immune response in a subject in need thereofagainst ExPEC serotypes O25B, and one or more additional E. coliO-antigens selected from the group consisting of E. coli O1A, O2 and O6Aantigens. The method comprises administering to a subject in needthereof an E. coli O25B antigen at a first effective amount of 5 to 18μg per administration, and E. coli O1A, O2 and O6A antigens each at aneffective amount that is independently 10% to 100% of the firsteffective amount, wherein each of the E. coli O25B antigen and theadditional E. coli O-antigens is independently covalently bound to anEPA carrier protein. In a preferred embodiment, each of the E. coli O1A,O2 and O6A antigens is independently administered at an effective amountof at least 3 μg per administration, more preferably, at an effectiveamount of at least 4 μg per administration.

In a method according to an embodiment of the invention, theadministered effective amount of E. coli O25B antigen is same or higherthan the administered effective amount of any of the additional Oantigens. For example, the E. coli O25B antigen can be administered at afirst effective amount of 4 to 24 μg per administration, and the atleast one additional E. coli O-antigen can be administered at a secondeffective amount that is, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% of the first effective amount. When more than one additionalO antigens are administered in combination, all of the additional Oantigens can be administered at the same effective amount that is10-100% of the first effective amount for the E. coli O25B antigen. Theadditional O antigens can also be administered at different effectiveamounts each of which is independently 10-100% of the first effectiveamount for the E. coli O25B antigen. Preferably, the E. coli O25Bantigen is administered at the first effective amount of 5 μg to 18 μg,and the at least one additional O antigen is administered at a secondeffective amount that is independently 50% to 100% of the firsteffective amount.

In one embodiment according to the invention, a method of inducing animmune response to ExPEC in a subject in need thereof comprisesadministering to the subject an E. coli O25B antigen at a firsteffective amount of, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16μg per administration, and E. coli O1A, O2 and O6A antigens each at aneffective amount that is independently, e.g., 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or 100% of the first effective amount, wherein eachof the E. coli O25B antigen and the additional E. coli O-antigens isindependently covalently bound to an EPA carrier protein.

In one preferred embodiment, the invention relates to a method ofinducing an immune response to ExPEC in a subject in need thereof,comprising administering to the subject an E. coli O25B antigen at afirst effective amount of 5 to 18 μg per administration, and a secondeffective amount of each of an E. coli O1A antigen, an E. coli O2antigen and an E. coli O6A antigen, wherein each of the E. coli O25B,O1A, O2 and O6A antigens are independently covalently bound to an EPAcarrier protein, and the ratio of the first effective amount to thesecond effective amount is 1:1 to 2:1. Preferably, the E. coli O25B,O1A, O2 and O6A antigens are administered to the subject at a dosageratio of 1:1:1:1 or 2:1:1:1, and the E. coli O25B antigen isadministered at 5 μg, 8 μg or 16 μg per administration. Also preferably,the E. coli O25B, O1A, O2 and O6A antigens are administered to thesubject in one composition.

Provided herein are methods and uses of compositions of the inventionfor inducing an immune response to ExPEC in a subject in need thereof,comprising administering to the subject an E. coli O25B antigencovalently bound to an EPA carrier protein, and at least one additionalE. coli O antigen covalently bound to the EPA carrier protein. In aspecific embodiment, the compositions described herein are used tovaccinate a human subject to induce a protective immunity against ExPECinfection of the human subject.

Further provided herein are methods of inducing the production ofopsonophagocytic antibodies against ExPEC in a subject in need thereof,comprising administering to the subject an E. coli O25B antigencovalently bound to an EPA carrier protein, and at least one additionalE. coli O antigen covalently bound to the EPA carrier protein.

In one embodiment, said subject has an ExPEC infection at the time ofadministration. In another embodiment, said subject does not have anExPEC infection at the time of administration. Examples of infectionscaused by ExPEC include, but are not limited to, urinary tractinfection, surgical-site infection, bacteremia, abdominal or pelvicinfection, pneumonia, nosocomial pneumonia, osteomyelitis, cellulitis,wound infection, meningitis, neonatal meningitis, peritonitis,cholangitis, soft-tissue infections, pyomyositis, septic arthritis, andsepsis. Preferably, the infection caused by ExPEC is an invasive ExPECdisease caused by ExPEC serotypes of which antigens are included in thecompositions or methods according to embodiments of the invention.

The methods of inducing an immune response in a subject described hereinresult in vaccination of the subject against infection by the ExPECstrains whose O-antigens are present in the composition(s). When anO-antigen subtype is used, a method of the invention can also induceimmune response to another O-antigen subtype having similarantigenicity.

In a specific embodiment, the immune response induced by a method orcomposition described herein is effective to prevent and/or treat aninfection caused by E. coli of the O25 serotype. In a specificembodiment, said O25 serotype is O25B. In another specific embodiment,said O25 serotype is O25A.

In a specific embodiment, the immune response induced by a method orcomposition described herein is effective to prevent and/or treat aninfection caused by E. coli of the O25 serotype, e.g. O25B serotype, andO1 serotype, e.g. O1A serotype.

In a specific embodiment, the immune response induced by a method orcomposition described herein is effective to prevent and/or treat aninfection caused by E. coli of the O25 serotype, e.g. O25B serotype, andO2 serotype.

In a specific embodiment, the immune response induced by a method orcomposition described herein is effective to prevent and/or treat aninfection caused by E. coli of the O25 serotype, e.g. O25B serotype, andO6 serotype, e.g. O6A serotype.

In a specific embodiment, the immune response induced by a method orcomposition described herein is effective to prevent and/or treat aninfection caused by E. coli of the O25 serotype (e.g. O25B and/or O25A),and two or more of the following E. coli serotypes: O1 (e.g., O1A, O1B,and/or O1C), O2, and/or O6 (e.g., O6A and/or O6GlcNAc).

In a specific embodiment, the immune response induced by a method orcomposition described herein is effective to prevent and/or treat aninfection caused by each of the following E. coli serotypes: O25 (e.g.,O25B and/or O25A), O1 (e.g., O1A, O1B, and/or O1C), O2, and O6 (e.g.,O6A and/or O6GlcNAc).

In a specific embodiment, the immune response induced by a method orcomposition described herein is effective to prevent and/or treat aninfection caused by E. coli of the O25 serotype, e.g. O25B serotype, andan E. coli serotype other than O1, O2, O6, or O25, including, but notlimited to, the additional O serotypes listed in Tables 1A-1C.

In order to immunize a subject against an ExPEC infection, the subjectcan be administered a single composition described herein, wherein saidcomposition comprises E. coli O25B antigen, and one, two, three, four,or more additional E. coli O antigens described herein, each covalentlybound to an EPA carrier protein. Alternatively, in order to treat asubject having an ExPEC infection or immunize a subject against an ExPECinfection, the subject can be administered multiple compositionsdescribed herein in combination. For example, a subject can beadministered a composition comprising E. coli O25B antigen conjugated toan EPA carrier protein, in combination with the administration of two,three, four, or more compositions comprising additional O antigenconjugates according to embodiments of the invention.

In certain embodiments, the immune response induced in a subjectfollowing administration of a composition described herein is effectiveto prevent or reduce a symptom resulting from an ExPEC infection,preferably in at least 30%, more preferably at least 40%, such as atleast 50%, of the subjects administered with the composition. Symptomsof ExPEC infection may vary depending on the nature of the infection andmay include, but are not limited to: dysuria, increased urinaryfrequency or urgency, pyuria, hematuria, back pain, pelvic pain, painwhile urinating, fever, chills, and/or nausea (e.g., in subjects havinga urinary tract infection caused by ExPEC); high fever, headache, stiffneck, nausea, vomiting, seizures, sleepiness, and/or light sensitivity(e.g., in subjects having meningitis caused by ExPEC); fever, increasedheart rate, increased respiratory rate, decreased urine output,decreased platelet count, abdominal pain, difficulty breathing, and/orabnormal heart function (e.g., in subjects having sepsis caused byExPEC).

In certain embodiments, the immune response induced in a subjectfollowing administration of a composition described herein is effectiveto reduce the likelihood of hospitalization of a subject suffering froman ExPEC infection. In some embodiments, the immune response induced ina subject following administration of a composition described herein iseffective to reduce the duration of hospitalization of a subjectsuffering from an ExPEC infection.

E. coli O-Antigens

Embodiments of the invention relate to compositions and methods relatingto E. coli O25B antigen and one or more additional E. coli O antigens.Preferably, the additional O antigen is prevalent among the clinicalisolates of E. coli. Examples of E. coli antigens that can be used inthe invention include, but are not limited to, the E. coli O25B, O1A,O2, and O6A antigens.

As used herein an “E. coli O25B antigen” refers to an O antigen specificto the E. coli O25B serotype. In one embodiment, an E. coli O25B antigencomprises the structure of Formula O25B:

preferably, the E. coli O25B antigen comprises the structure of FormulaO25B′ :

wherein the n in Formula O25B or Formula O25B′ is an integer of 1 to 30,1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In oneembodiment of the invention, the n in Formula O25B or Formula O25B′ isan integer of 10-20.

Preferably, a population of E. coli O25B antigens having the structureof Formula O25B, more preferably Formula O25B′, is used in compositionsand methods according to embodiments of the invention, wherein the n ofat least 80% of the E. coli O25B antigens in the population is aninteger of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20,or 15 to 20. In one embodiment of the invention, the n of at least 80%of the E. coli O25B antigens in the population is an integer of 10-20.

As used herein, an “E. coli O1 antigen” refers to an O antigen specificto the E. coli O1 serotype. In one embodiment, an E. coli O1 antigen isan E. coli O1A antigen.

As used herein, an “E. coli O1A antigen” refers to an O antigen specificto the E. coli O1A serotype. In one embodiment, an E. coli O1A antigencomprises the structure of Formula O1A:

preferably, the E. coli O1A antigen comprises the structure of FormulaO1A′:

wherein the n in Formula O1A or Formula O1A′ is an integer of 1 to 30, 1to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30,5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In oneembodiment, the n in Formula O1A or Formula O1A′ is an integer of 7-15.

Preferably, a population of E. coli O1A antigens having the structure ofFormula O1A, more preferably Formula O1A′, is used in compositions andmethods according to embodiments of the invention, wherein the n of atleast 80% of the E. coli O1A antigens in the population is of 1 to 30, 1to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30,5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In oneembodiment, then of at least 80% of the E. coli O1A antigens in thepopulation is an integer of 5-15.

As used herein, an “E. coli O2 antigen” refers to an O antigen specificto the E. coli O2 serotype. In one embodiment, an E. coli O2 antigencomprises the structure of Formula O2:

preferably, the E. coli O2 antigen comprises the structure of FormulaO2′:

wherein the n in Formula O2 or Formula O2′ is an integer of 1 to 30, 1to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30,5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In oneembodiment, the n in Formula O2 or Formula O2′ is an integer of 8-16.

Preferably, a population of E. coli O2 antigens having the structure ofFormula O2, more preferably Formula O2′, is used in compositions andmethods according to embodiments of the invention, wherein the n of atleast 80% of the E. coli O2 antigens in the population is of 1 to 30, 1to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30,5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In oneembodiment, then of at least 80% of the E. coli O2 antigens in thepopulation is an integer of 5-20.

As used herein, an “E. coli O6 antigen” refers to an O antigen specificto the E. coli O6 serotype. In one embodiment, an E. coli O6 antigen isan E. coli O6A.

As used herein, an “E. coli O6A antigen,” also referred to as “E. coliO6K2 antigen” or “E. coli O6Glc antigen,” refers to an O antigenspecific to the E. coli O6A serotype. In one embodiment, an E. coli O6Aantigen comprises the structure of Formula O6A:

preferably, the E. coli O6A antigen comprises the structure of FormulaO6A′:

wherein the β1, 2 linkage is also named β2 linkage, the n in Formula O6Aor Formula O6A′ is an integer of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment, the n in FormulaO6A or Formula O6A′ is an integer of 8-18.

Preferably, a population of E. coli O6A antigens having the structure ofFormula O6A, more preferably Formula O6A′, is used in compositions andmethods according to embodiments of the invention, wherein the n of atleast 80% of the E. coli O6A antigens in the population is of 1 to 30, 1to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30,5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In oneembodiment, the n of at least 80% of the E. coli O6A antigens in thepopulation is an integer of 5-20.

In a preferred embodiment, a composition of the invention comprises E.coli O25B antigens having the structure of formula O25B′, wherein the nof at least 80% of the E. coli O25B antigens in the population is aninteger of 10-20; E. coli O1A antigens having the structure of formulaO1A′, wherein the n of at least 80% of the E. coli O1A antigens in thepopulation is an integer of 5-15; E. coli O2 antigens having thestructure of formula O2′, wherein the n of at least 80% of the E. coliO2 antigens in the population is an integer of 5-20; and E. coli O6Aantigens having the structure of formula O6A′, wherein the n of at least80% of the E. coli O6A antigens in the population is an integer of 5-20,wherein each of the O-antigens is covalently bound to an EPA carrierprotein having the amino acid sequence of SEQ ID NO:1.

An E. coli O antigen useful in the invention can be produced by methodsknown in the art in view of the present disclosure. For example, theycan be produced from a cell, preferably a recombinant cell that isoptimized for the biosynthesis of the O antigen. See, e.g., relevantdisclosure on the nucleic acids, proteins, host cells, productionmethods, etc., for E. coli O antigen biosynthesis in WO 2006/119987, WO2009/104074, International Patent Application No. PCT/EP2015/053739,Ihssen et al., 2010, Microbial Cell Factories 9, 61, the disclosures ofwhich are herein incorporated by reference in their entirety.

EPA Carrier Protein

According to embodiments of the invention, each E. coli O antigen iscovalently bound to an EPA carrier protein (see, e.g., Ihssen et al.,2010, Microbial Cell Factories 9, 61). Various detoxified EPA variantshave been described in literature and can be used as EPA carrierproteins in the conjugates described herein.

In certain embodiments, the EPA carrier proteins used in the conjugatesdescribed herein are EPAs modified in such a way that the protein isless toxic and/or more susceptible to glycosylation. For example,detoxification can be achieved by mutating and deleting thecatalytically essential residues, such as L552V and ΔE553, according toLukac et al., Infect Immun, 56: 3095-3098, 1988 and Ho et al., HumVaccin, 2:89-98, 2006. In a specific embodiment, the carrier proteinsused in the generation of the conjugates described herein are EPAsmodified such that the number of glycosylation sites in the carrierproteins is optimized in a manner that allows for lower concentrationsof the protein to be administered, e.g., in an immunogenic composition,in its bioconjugate form.

In certain embodiments, the EPA carrier proteins are EPAs modified toinclude 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosylation sites thanwould normally be associated with the carrier protein (e.g., relative tothe number of glycosylation sites associated with the carrier protein inits native/natural, e.g., “wild-type” state). In specific embodiments,introduction of glycosylation sites is accomplished by insertion ofglycosylation consensus sequences (e.g., Asn-X-Ser(Thr), wherein X canbe any amino acid except Pro (SEQ ID NO: 2); or preferablyAsp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selectedfrom any natural amino acid except Pro (SEQ ID NO:3) (see WO2006/119987)) anywhere in the primary structure of the EPA protein. Inone particular embodiment, the EPA carrier protein comprises 4 consensusglycosylation sequences Asp/Glu-X-Asn-Z-Ser/Thr (SEQ ID NO: 3), and hasan amino acid sequence as provided in SEQ ID NO: 1.

In certain embodiments, the EPA carrier protein can be produced togetherwith a signal sequence (such as a signal peptide for E. coli DsbA, E.coli outer membrane porin A (OmpA), E. coli maltose binding protein(MalE), etc.) that targets the carrier protein to the periplasmic spaceof the host cell that expresses the carrier protein. The EPA carrierprotein can also be modified to a “tag,” i.e., a sequence of amino acidsthat allows for the isolation and/or identification of the carrierprotein.

An EPA carrier protein useful in the invention can be produced bymethods known in the art in view of the present disclosure. See, e.g.,relevant disclosure in e.g., Ihssen et al., 2010, Microbial CellFactories 9, 61, and in WO 2006/119987, WO 2009/104074, andInternational Patent application No. PCT/ EP2015/053739, the disclosureof which are herein incorporated by reference in their entirety.

Conjugates

In certain embodiments, a host cell can produce an E. coli O antigen andan EPA carrier protein, and covalently bind the O antigen to the EPAcarrier protein to form a bioconjugate useful in the invention. See,e.g., relevant disclosure in e.g., Ihssen et al., 2010, Microbial CellFactories 9, 61, and in WO 2006/119987, WO 2009/104074, andInternational Patent application No. PCT/ EP2015/053739, the disclosuresof which are herein incorporated by reference in their entirety.

Alternatively, the glycoconjugates can be prepared by chemicalsynthesis, i.e., prepared outside of host cells (in vitro). For example,the E. coli O-antigens described herein, e.g., O25B antigen, can beconjugated to carrier proteins using methods known to those of skill inthe art, including by means of using activation reactive groups in thepolysaccharide/oligosaccharide as well as the protein carrier. See,e.g., Pawlowski et al., 2000, Vaccine 18:1873-1885; and Robbins et al.,2009, Proc Natl Acad Sci USA 106:7974-7978, the disclosures of which areherein incorporated by reference. Such approaches comprise extraction ofantigenic polysaccharides/oligosaccharides from host cells, purifyingthe polysaccharides/oligosaccharides, chemically activating thepolysaccharides/oligosaccharides, and conjugating the polysaccharides/oligosaccharides to a carrier protein.

Bioconjugates have advantageous properties over glycoconjugates made invitro, e.g., bioconjugates require less chemicals in manufacture and aremore consistent and homogenous in terms of the final product generated.Thus, bioconjugates are preferred over chemically producedglycoconjugates.

In a specific embodiment, the EPA carrier protein is N-linked to an E.coli O-antigen useful in the invention. For example, the E. coli Oantigen is linked to the Asn residue in a glycosylation sequence of acarrier protein, such as Asn-X-Ser(Thr), wherein X can be any amino acidexcept Pro (SEQ ID NO: 2), preferably Asp(Glu)-X-Asn-Z-Ser(Thr), whereinX and Z are independently selected from any natural amino acid exceptPro (SEQ ID NO: 3).

The conjugates described herein can be purified by any method known inthe art for purification of a protein, for example, by chromatography(e.g., ion exchange, anionic exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. See, e.g.,Saraswat et al., 2013, Biomed. Res. Int. ID #312709 (p. 1-18); see alsothe methods described in WO 2009/104074. The actual conditions used topurify a particular conjugate will depend, in part, on the synthesisstrategy (e.g., synthetic production vs. recombinant production) and onfactors such as net charge, hydrophobicity, and/or hydrophilicity of thebioconjugate, and will be apparent to those having skill in the art.

Combination Therapies

In certain embodiments, a composition described herein is administeredto a subject in combination with one or more other therapies (e.g.,antibacterial or immunomodulatory therapies). The one or more othertherapies can be beneficial in the treatment or prevention of an ExPECinfection or can ameliorate a symptom or condition associated with anExPEC infection.

In some embodiments, the one or more other therapies are pain relieversor anti-fever medications. In certain embodiments, the therapies areadministered less than 5 minutes apart to less than 1 week apart. Anyanti-bacterial agents known to one of skill in the art (e.g.antibiotics) may be used in combination with a composition describedherein.

Patient Populations

In certain embodiments, a composition (or method) described herein isadministered (or applied) to a naive subject, i.e., a subject that doesnot have an ExPEC infection or has not previously had an ExPECinfection. In one embodiment, a composition (or method) described hereinis administered (or applied) to a naive subject that is at risk ofacquiring an ExPEC infection.

In certain embodiments, a composition (or method) described herein isadministered (or applied) to a subject who has been or was previouslydiagnosed with an ExPEC infection. In some embodiments, a composition(or method) described herein is administered (or applied) to a subjectinfected with ExPEC before symptoms manifest or symptoms become severe(e.g., before the patient requires hospitalization).

In certain embodiments, a composition (or method) described herein isadministered (or applied) to a subject who has been diagnosed with anuropathogenic E. coli (UPEC) infection. In some embodiments, acomposition (or method) described herein is administered (or applied) toa subject suffering from reoccurring urinary tract infections. In someembodiments, a composition (or method) described herein is administered(or applied) to a subject suffering from reoccurring urinary tractinfections, but is healthy at the moment of treatment. In someembodiments, a composition (or method) described herein is administered(or applied) to a subject having or at risk of acquiring bacteremia orsepsis.

In some embodiments, a subject to be administered (or applied) acomposition (or method) described herein is an animal. In certainembodiments, the animal is a canine. In certain embodiments, the animalis a feline. In certain embodiments, the animal is a horse. In certainembodiments, the animal is a cow. In certain embodiments, the animal isa mammal, e.g., a horse, swine, rabbit, mouse, or primate. In apreferred embodiment, the subject is a human.

In certain embodiments, a subject to be administered (or applied) acomposition (or method) described herein is a human subject, preferably,a human subject at risk of having an invasive ExPEC disease. In certainembodiments, a subject to be administered (or applied) a composition (ormethod) described herein is a human adult more than 50 years old. Incertain embodiments, a subject to be administered (or applied) acomposition (or method) described herein is a human adult more than 55,more than 60 or more than 65 years old.

In certain embodiments, a subject to be administered (or applied) acomposition (or method) described herein is a human child. In certainembodiments, a subject to be administered (or applied) a composition (ormethod) described herein is a human child. In certain embodiments, asubject to be administered (or applied) a composition (or method)described herein is a human infant, including a premature human infant.In some embodiments, a subject to be administered (or applied) acomposition (or method) described herein is a human toddler. In certainembodiments, a subject to be administered (or applied) a composition (ormethod) described herein is not an infant of less than 6 months old.

In certain embodiments, a subject to be administered (or applied) acomposition (or method) described herein is an individual who ispregnant. In certain embodiments, a subject to be administered (orapplied) a composition (or method) described herein is a woman who hasgiven birth 1, 2, 3, 4, 5, 6, 7, or 8 weeks earlier.

In certain embodiments, a subject to be administered (or applied) acomposition (or method) described herein is an individual at increasedrisk of ExPEC, e.g., an immunocompromised or immunodeficient individual,an individual scheduled for surgery or recently undergone a surgery, anindividual having a wound injury, an intensive care unit (ICU) orcritical care unit (CCU) patient, etc. In certain embodiments, a subjectto be administered (or applied) a composition (or method) describedherein is an individual in close contact with an individual having or atincreased risk of ExPEC infection.

In certain embodiments, a subject to be administered (or applied) acomposition (or method) described herein is a health care worker. Incertain embodiments, a subject to be administered (or applied) acomposition (or method) described herein is immunocompromised (e.g.,suffers from HIV infection) or immunosuppressed.

In certain embodiments, a subject to be administered (or applied) acomposition (or method) described herein has diabetes. In certainembodiments, a subject to be administered (or applied) a composition (ormethod) described herein has multiple sclerosis.

In certain embodiments, a subject to be administered (or applied) acomposition (or method) described herein has a condition that requiresthem to use a catheter, such as a urinary catheter. In certainembodiments, a subject to be administered (or applied) a composition (ormethod) described herein has a spinal cord injury.

In certain embodiments, the subject is a male who will undergo or hasrecently undergone a prostate biopsy.

In a preferred embodiment, the subject to be administered (or applied) acomposition (or method) described herein is an at-risk human adult inneed of immunization for the prevention of invasive ExPEC disease causedby ExPEC serotypes O1A, O2, O6A and O25B. Examples of at-risk humaninclude, but are not limited to, those described herein supra. Otherexamples of at-risk human include, e.g., individuals having transrectalultrasonography with prostate needle biopsy (TRUS-PNB) or recurrenturosepsis, residents of a long term care facility (LTCF), long term care(LTAC)-assisted living, pre-surgery patients (including but not limitedto, patients scheduled for genito-urinary/abdominal surgery);pre-dialysis patients, pre-dialysis, etc.

Dosage and Frequency of Administration

Administration of the conjugates of O-antigens and an EPA carrierprotein and/or composition thereof can be done via various routes knownto the clinician, for instance subcutaneous, parenteral, intravenous,intramuscular, topical, oral, intradermal, transdermal, intranasal, etc.In one embodiment, administration is via intramuscular injection.

According to embodiments of the invention, the dosage level of E. coliO25B antigen covalently should be no less, preferably more, than thedosage levels of the other E. coli O antigens used in a composition ormethod of the invention, wherein each of the E. coli O antigens iscovalently bound to an EPA carrier protein. The precise dosage to beemployed in the formulation and method will depend on the route ofadministration, and the seriousness of the infection, and should bedecided according to the judgment of the practitioner and each subject'scircumstances.

In certain embodiments of the invention, exemplary dosages for E. coliO25B antigen range from 4 to 24 μg of O25B antigen per administration,and the exemplary dosages for each of the additional E. coli O antigensto be used in combination with the E. coli O25B antigen range from 10%to 100% of the dosage of E. coli O25B antigen, wherein each of the E.coli O antigens is covalently bound to an EPA carrier protein. Incertain embodiments, an exemplary dosage for an E. coli O25Bglycoconjugate is, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 or 24 μg of O25B antigen per administration,and an exemplary dosage for another E. coli O glycoconjugate to be usedin combination with the E. coli O25B glycoconjugate is, e.g., 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the dosage for the E. coliO25B glycoconjugate, wherein the dosage is calculated by the amount ofthe O antigen in the O glycoconjugates per administration.

In certain embodiments of the invention, a subject in need thereof isadministered with 0.5 ml of a composition according to the invention.

In certain embodiments, an exemplary dosage for per administration to ahuman subject corresponds to 0.5 ml of a composition containing a firstconcentration of about 8-48 μg/mL, e.g., about 8, 12, 16, 20, 24, 28,32, 36, 40, 44 or 48 μg/mL, of E. coli O25B antigen covalently bound toan EPA carrier protein, and a concentration of 10% to 100% of the firstconcentration of one or more additional E. coli O antigens covalentlybound to the EPA carrier protein.

In certain embodiments, an exemplary dosage for per administration to ahuman subject corresponds to 0.5 ml of a composition containing aconcentration of about 16 μg/mL of E. coli O25B antigen covalently boundto an EPA carrier protein, about 8 μg/mL of E. coli O1A antigencovalently bound to an EPA carrier protein, about 8 μg/mL of E. coli O2antigen covalently bound to an EPA carrier protein, and about 8 μg/mL ofE. coli O6A antigen covalently bound to an EPA carrier protein.

In certain embodiments, an exemplary dosage for per administration to ahuman subject corresponds to 0.5 ml of a composition containing aconcentration of about 16 μg/mL of E. coli O25B antigen covalently boundto an EPA carrier protein, about 16 μg/mL of E. coli O1A antigencovalently bound to an EPA carrier protein, about 16 μg/mL of E. coli O2antigen covalently bound to an EPA carrier protein, and about 16 μg/mLof E. coli O6A antigen covalently bound to an EPA carrier protein.

In certain embodiments, an exemplary dosage for per administration to ahuman subject corresponds to 0.5 ml of a composition containing aconcentration of about 32 μg/mL of E. coli O25B antigen covalently boundto an EPA carrier protein, about 16 μg/mL of E. coli O1A antigencovalently bound to an EPA carrier protein, about 16 μg/mL of E. coli O2antigen covalently bound to an EPA carrier protein, and about 16 μg/mLof E. coli O6A antigen covalently bound to an EPA carrier protein.

In certain embodiments, an exemplary dosage for per administration to ahuman subject corresponds to 0.5 ml of a composition containing aconcentration of about 32 μg/mL of E. coli O25B antigen covalently boundto an EPA carrier protein, about 32 μg/mL of E. coli O1A antigencovalently bound to an EPA carrier protein, about 32 μg/mL of E. coli O2antigen covalently bound to an EPA carrier protein, and about 32 μg/mLof E. coli O6A antigen covalently bound to an EPA carrier protein.

In certain embodiments, E. coli O-antigen conjugates, preferablybioconjugates, described herein or a composition described herein isadministered to a subject once as a single dose. In certain embodiments,E. coli O-antigen conjugates, preferably bioconjugates, described hereinor a composition described herein is administered to a subject as asingle dose followed by a second dose 3 to 6 weeks later. In accordancewith these embodiments, booster inoculations can be administered to thesubject at 6 to 24 month intervals following the second inoculation. Incertain embodiments, the booster inoculations can utilize a different E.coli O-antigen, bioconjugate, or composition. In some embodiments, theadministration of the same E. coli O-antigen conjugate, or compositioncan be repeated and the administrations can be separated by at least 1day, 2 days, 3 days, 5 days, 7 days, 10 days, 15 days, 30 days, 45 days,2 months, 75 days, 3 months, or at least 6 months. In certainembodiments, an E. coli O-antigen conjugate described herein or acomposition described herein is administered to a subject as a singledose once per year. In certain embodiments, an E. coli O-antigenconjugate described herein or a composition described herein isadministered to a subject as a single dose once per n years, n being forinstance about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years.

In certain embodiments, an E. coli O-antigen conjugate described hereinor a composition described herein is administered to a subject as 2, 3,4, 5 or more doses 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks apart.In some embodiments, 2, 3, 4, 5 or more doses of an E. coli O-antigenconjugate described herein or a composition described herein areadministered to a subject 2, 3, 4, 5 or 6 weeks apart. In certainembodiments, the E. coli O-antigen conjugate, or compositionadministered is the same each time. In certain embodiments, the E. coliO-antigen conjugate, or composition administered is different each time.

Assays

The ability of the conjugates/compositions described herein to generatean immune response in a subject can be assessed using any approach knownto those of skill in the art in view of the present disclosure.

Assay for Assessing Ability of Bioconjugates to Induce an ImmuneResponse

In some embodiments, the ability of a bioconjugate to generate an immuneresponse in a subject can be assessed by immunizing a subject (e.g., amouse) or set of subjects with the bioconjugate and immunizing anadditional subject (e.g., a mouse) or set of subjects with a control(e.g., a placebo). The subjects or set of subjects can subsequently bechallenged with ExPEC and the ability of the ExPEC to cause disease(e.g., UTI) in the subjects or set of subjects can be determined. Thoseskilled in the art will recognize that if the subject or set of subjectsimmunized with the control suffer(s) from disease subsequent tochallenge with the ExPEC but the subject or set of subjects immunizedwith a bioconjugate(s) or composition thereof described herein sufferless from or do not suffer from disease, then the bioconjugate is ableto generate an immune response in a subject. The ability of abioconjugate(s) or composition thereof described herein to induceantiserum that cross-reacts with an O-antigen from ExPEC can be testedby, e.g., an immunoassay, such as an ELISA.

In Vitro Bactericidal Assays

The ability of the conjugates/compositions described herein to generatean immune response in a subject can be assessed using a serumbactericidal assay (SBA) or opsonophagocytotic killing assay (OPK),which represents an established and accepted method that has been usedto obtain approval of glycoconjugate-based vaccines. Such assays arewell-known in the art and, briefly, comprise the steps of generating andisolating antibodies against a target of interest (e.g., an O-antigen,e.g., O25B, of E. coli) by administering to a subject (e.g., a mouse) acompound that elicits such antibodies. Subsequently, the bactericidalcapacity of the antibodies can be assessed by, e.g., culturing thebacteria in question (e.g., E. coli of the relevant serotype) in thepresence of said antibodies and complement and—depending on theassay—neutrophilic cells and assaying the ability of the antibodies tokill and/or neutralize the bacteria, e.g., using standardmicrobiological approaches.

KITS

Provided herein is a pack or kit comprising one or more containersfilled with one or more of the ingredients of the compositions describedherein, such as one or more E. coli O antigens and/or conjugates of theE. coli O antigens covalently bound to an EPA carrier protein accordingto embodiments of the invention. Optionally associated with suchcontainer(s) can be a notice or instructions in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Thekits encompassed herein can be used in the above methods of treatmentand immunization of subjects.

The following examples of the invention are to further illustrate thenature of the invention. It should be understood that the followingexamples do not limit the invention and that the scope of the inventionis to be determined by the appended claims.

EMBODIMENTS

Embodiment 1 is a composition comprising a first concentration of an E.coli O25B antigen polysaccharide, and a second concentration of each ofan E. coli O1A antigen polysaccharide, an E. coli O2 antigenpolysaccharide and an E. coli O6A antigen polysaccharide, wherein theratio of the first concentration to the second concentration is 1:1 to2:1, each of the E. coli O25B, O1A, O2 and O6A antigen polysaccharidesare independently covalently bound to a detoxified exotoxin A ofPseudomonas aeruginosa (EPA) carrier protein, and the firstconcentration is 10 to 36 μg/ml.

Embodiment 2 is the composition of embodiment 1, comprising the E. coliO25B, O1A, O2 and O6A antigen polysaccharides at a weight ratio of1:1:1:1.

Embodiment 3 is the composition of embodiment 1, comprising the O25B,O1A, O2 and O6A antigen polysaccharides at a weight ratio of 2:1:1:1.

Embodiment 4 is the composition of any one of embodiments 1 to 3,comprising 16 μg/ml of the O25B antigen polysaccharide.

Embodiment 5 is the composition of any one of embodiments 1 to 3,comprising 32 μg/ml of the O25B antigen polysaccharide.

Embodiment 6 is a multivalent immune composition comprising an E. coliO25B antigen polysaccharide at a first dose of 5 to 18 μg, and an E.coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharideand an E. coli O6A antigen polysaccharide each at a dose that isindependently 50% to 100% of the first dose, wherein each of the E. coliO25B, O1A, O2 and O6A antigen polysaccharides are independentlycovalently bound to a detoxified exotoxin A of Pseudomonas aeruginosa(EPA) carrier protein.

Embodiment 7 is a multivalent immune composition comprising an E. coliO25B antigen polysaccharide having the structure of Formula O25B′:

an E. coli O1A antigen polysaccharide having the structure of FormulaO1A′:

an E. coli O2 antigen polysaccharide having the structure of FormulaO2′:

and an E. coli O6A antigen polysaccharide having the structure ofFormula O6A′:

wherein n is independently an integer of 5 to 25, and each of the E.coli O25B, O1A, O2 and O6A antigen polysaccharides are independentlycovalently bound to a carrier protein having the amino acid sequence ofSEQ ID NO:1; and the concentrations of the E. coli O25B, O1A, O2, O6Aantigen polysaccharides in the compositions are respectively 16:8:8:8μg/ml, 16:16:16:16 μg/ml, 32:16:16:16 μg/ml or 32:32:32:32 μg/ml.

Embodiment 8 is a method of inducing an immune response toextra-intestinal pathogenic E. coli (ExPEC) in a subject in needthereof, comprising administering to the subject a composition of anyone of embodiments 1 to 7.

Embodiment 9 is a method of inducing an immune response toextra-intestinal pathogenic E. coli (ExPEC) in a subject in needthereof, comprising administering to the subject a first effectiveamount of an E. coli O25B antigen polysaccharide, and a second effectiveamount of each of an E. coli O1A antigen polysaccharide, an E. coli O2antigen polysaccharide and an E. coli O6A antigen polysaccharide,wherein the ratio of the first effective amount to the second effectiveamount is 1:1 to 2:1, each of the E. coli O25B, O1A, O2 and O6A antigenpolysaccharides are independently covalently bound to a detoxifiedexotoxin A of Pseudomonas aeruginosa (EPA) carrier protein, and thefirst effective amount is 5 to 18 μg per administration.

Embodiment 10 is the method of embodiment 9, wherein the E. coli O25B,O1A, O2 and O6A antigen polysaccharides are administered at a dosageratio of 1:1:1:1.

Embodiment 11 is the method of embodiment 9, wherein the E. coli O25B,O1A, O2 and O6A antigen polysaccharides are administered at a dosageratio of 2:1:1:1.

Embodiment 12 is the method of any one of embodiments 8 to 11, wherein 8μg of the O25B antigen polysaccharide is administered peradministration.

Embodiment 13 is the method of any one of embodiments 8 to 11, wherein16 μg of the O25B antigen polysaccharide is administered peradministration.

Embodiment 14 is the method of any one of embodiments 8 to 13, whereinthe E. coli O25B, O1A, O2 and O6A antigen polysaccharides areadministered together in one composition.

Embodiment 15 is a method of inducing an immune response toextra-intestinal pathogenic E. coli (ExPEC) in a subject in needthereof, comprising administering to the subject an E. coli O25B antigenpolysaccharide having the structure of Formula O25B′:

an E. coli O1A antigen polysaccharide having the structure of FormulaO1A′:

an E. coli O2 antigen polysaccharide having the structure of FormulaO2′:

and an E. coli O6A antigen polysaccharide having the structure ofFormula O6A′:

wherein n is independently an integer of 5 to 25, each of the E. coliO25B, O1A, O2 and O6A antigen polysaccharides are independentlycovalently bound to a carrier protein having the amino acid sequence ofSEQ ID NO:1, and the E. coli O25B, O1A, O2 and O6A antigenpolysaccharides are administered at 8:4:4:4 μg, 8:8:8:8 μg, 16:8:8:8 μgor 16:16:16:16 μg per administration.

Embodiment 16 is the method of any one of embodiments 8 to 15, whereinthe immune response limits the severity of or prevents an invasive ExPECdisease caused by ExPEC serotypes O1A, O2 and O6A and O25B in an at-riskhuman subject.

Embodiment 17 is the method of embodiment 16, wherein the adult humansubject has or is at risk of having an invasive ExPEC disease selectedfrom the group consisting of urinary tract infection, a surgical-siteinfection, an abdominal or pelvic infection, pneumonia, nosocomialpneumonia, osteomyelitis, cellulitis, sepsis, bacteremia, a woundinfection, pyelonephritis, meningitis, neonatal meningitis, peritonitis,cholangitis, soft-tissue infections, pyomyositis and septic arthritis.

Embodiment 18 is a process of making a composition of any one ofembodiments 1 to 7, comprising combining the E. coli O25B antigenpolysaccharide, the E. coli O1A antigen polysaccharide, the E. coli O2antigen polysaccharide and the E. coli O6A antigen polysaccharide tothereby obtain the composition.

EXAMPLES O-Antigen Bioconjugates

O1A-EPA, O2-EPA, O6A-EPA and O25B-EPA bioconjugates containing,respectively, E. coli O1A, O2, O6A and O25B covalently linked to theglycosylation sites of an EPA protein carrier can be produced, purified,and characterized as described in, e.g., Ihssen et al., 2010, MicrobialCell Factories 9, 61, and in WO 2006/119987, WO 2009/104074, andInternational Patent application No. PCT/ EP2015/053739, the disclosureof which are herein incorporated by reference in their entirety. Thebioconjugates are synthesized using recombinant E. coli cells, whichexpress the polysaccharide-synthesizing enzymes of the differentO-serotypes in the presence of oligosaccharyltransferase Pg1B, and aprotein carrier (EPA). In this approach, the glycoconjugate vaccine canbe expressed in the periplasm of E. coli, extracted and purified througha biochemical process illustrated in FIG. 1 and FIG. 2 . Table 2indicates host strains used for the production of conjugates accordingto an embodiment of the invention.

TABLE 2 Host strains for production of preclinical, toxicology study andclinical batches EPA expression PglB expression Product Strain plasmidplasmid EPA-O1A W3110 Δrfb::rfb(upec032) ΔwaaL pGVXN1076 pGVXN970 EPA-O2W3110 Δrfb::rfb(upec116) ΔwaaL pGVXN1076 pGVXN971 EPA-O6A W3110Δrfb::rfb(CCUG11309) ΔwaaL pGVXN659 pGVXN114 EPA-O25B W3110Δrfb::rfb(upec138) ΔwaaL ΔgtrABS pGVXN1076 pGVXN970

For example, for O25B-EPA production, a strain with a genomicallyintegrated O25B cluster was constructed: W3110 ΔwaaL ΔgtrABSΔrfbO16::rfb(upec138), which was transformed with plasmids pGVXN1076(which expresses the EPA having the amino acid sequence of SEQ ID NO:1)and pGVXN970 (which expressed the oligosaccharyl transferase PelB)(WO/2009/104074). This strain was constructed starting from strain W3110by the methods of Datsenko and Wanner (2000, Proc Natl Acad Sci USA 97:6640-6645) and a homologous recombination technique for site directedintegration of large inserts into bacterial chromosomes (see WO2014/057109). The rfb cluster related to the O25B antigen was clonedfrom E. coli strain upec138, which is positive for O25B. The recombinanthost cells produced O25B/EPA bioconjugates in the periplasm. Theresulting O25B bioconjugates were characterized using standard releaseand characterization assays. Bioconjugates were purified using twoconsecutive anionic exchange and size exclusion chromatography steps,yielding 98.1% pure O25B bioconjugate preparations.

Similarly, host strains for recombinant production of O1A-EPA, O2-EPA,and O6A-EPA were constructed (Table 2). These strains include the rfbclusters related to O1A, O2 and O6A cloned from E. coli strain upec032,upec116 and CCUG11309, respectively. Bioconjugates of O1A-EPA, O2-EPA,and O6A-EPA were produced from these recombinant host cells, andpurified using methods known in the art in view of the presentdisclosure.

SDS PAGE quantification was used for purity analysis. Sugar to proteinratios were calculated based on sugar quantification by the anthroneassay (see Laurentin and Edwards, 2003, Anal Biochem 315, 143-145) andthe BCA assay for protein concentration. Analytical size exclusionchromatography showed a monomeric state of the particles in agreementwith the expected hydrodynamic radius of EPA with attached glycanchains.

The bioconjugates and an un-glycosylated EPA reference standard wereanalyzed by size-exclusion chromatography with multi-angle lightscattering (SEC-MALS), in order to quantify the degree of mono- anddi-glycosylation of the individual bioconjugates, and to determine themolecular mass (MW) of the protein carrier and of the O-PS attached toit. The samples were separated on a TSKgel-G3000 SWxl column inphosphate buffer (pH 7.0; 50 mM NaCl, 150 mM sodium phosphate) andmonitored by UV (214 and 280 nm), refractive index (RI) and multi anglelight scattering (MALS).

Vaccine Compositions

This Example illustrates the vaccine compositions useful for theinvention.

TABLE 3-1 Vaccine Compositions Ingredient Amount (μg/mL) Activesubstance ExPEC4V Composition 3 O-antigen polysaccharide E. coli O1A 8 8E. coli O2 8 8 E. coli O6A 8 8 E. coli O25B 8 16 Carrier Protein EPA 109Expected: 126 Excipients TBS buffer, containing pH 7.4 Tris 25 mM NaCl137 mM KCl 2.7 mM

TABLE 3-2 Vaccine Compositions Ingredient Amount (ug/mL) Activesubstance Composition 1 Composition 2 O-antigen polysaccharide E. coliO1A 32 16 E. coli O2 32 16 E. coli O6A 32 16 E. coli O25B 32 32 CarrierProtein EPA Expected: 436 Expected: 251 Excipients TBS buffer,containing pH 7.4 Tris 25 mM NaCl 137 mM KCl 2.7 mM

Each of the above illustrated liquid vaccine compositions is packaged ina vial ready for injection. The vaccine products should be stored at +2°C. to +8° C.

The active substances in the vaccine composition are glycosylatedproteins (bioconjugates composed of the EPA protein carrier covalentlylinked to an E. coli O antigen polysaccharide) and the dose iscalculated based on the content of the polysaccharide moiety (O antigen)only.

The dose of the EPA carrier protein depends on thepolysaccharide-to-protein ratio. The estimated polysaccharide-to-proteinratio was between 15% and 50% depending on the O-antigen serotypes,i.e., the weight of polysaccharide in a conjugate is about 15% to 50% ofthe weight of the EPA protein carrier in the conjugate. For eachserotype in ExPEC4V, the polysaccharide-to-protein ratio was quantified,e.g., the amount of polysaccharide (O-antigen) was measured by theanthrone assay (see Laurentin and Edwards, 2003, Anal Biochem 315,143-145) and the bicinchoninic acid (BCA) assay was used to measure theprotein concentration. For each serotype in Products 1-3, the value ofEPA provided in Tables 3-1 and 3-2 is the expected value based on theanalytical results from ExPEC4V.

Stability of the tetravalent vaccine compositions (O25B, O1A, O2 and O6bioconjugates) was tested during over a 3 month period. The studiesincluded accelerated and stress storage conditions to identifydegradation pathways. These studies demonstrate that the activesubstances and the tetravalent vaccine compositions are stable for atleast three months, and thus are suitable vaccine compositions withrespect to stability.

Repeated Dose Toxicity Study in Rats

A good laboratory practice (GLP) toxicity study with a 14-day recoveryperiod was conducted in Sprague-Dawley rats to assess the toxicity andlocal tolerance of a vaccine composition following 2 intramuscular(i.m.) injections (in quadriceps femoris muscle) on Days 1 and 14.Reversibility, persistence, and delayed occurrence of any changes wereassessed after a 14-day recovery period (i.e., on Day 28). The design ofthe Phase 1-enabling repeated dose toxicity study in the rat is outlinedin Table 4.

TABLE 4 Design of Repeated Dose Toxicity Study in the Rat Dose MainRecovery Concentration volume Animals Animals Group Dose level (μg/mL)(mL)^(b) (n)^(c) (n)^(d) Vehicle^(a) — — 0.5 10M + 10 F 5M + 5 F ExPEC4V4 μg polysaccharide per polysaccharide per 0.5 10M + 10 F 5M + 5 FO-antigen = total of 16 μg O-antigen: 8 μg/ polysaccharide per dose +mL + protein carrier 48 μg per protein EPA: 96 μg/mL carrier EPA doseEPA = ExoProtein A/Pseudomonas aeruginosa exotoxin A, detoxified formused as protein carrier; F = female, M = male Note: Day 1 = start oftreatment ^(a)The vehicle group was administered the formulation buffer(vehicle control) ^(b)Animals received 2 injections of 0.25 mL perdosing occasion (left and right hind leg). Animals were dosed on 2occasions: Day 1 and Day 14 ^(c)Main animals were euthanized on Day 17^(d)Recovery animals were euthanized on Day 28, ie 14 days after thesecond treatment

A dose of 4 μg per O-antigen polysaccharide (PS) of ExPEC4V (total PSdose of 16 μg) was tested in this study. This dose is equivalent to themaximum dose that was evaluated in the Phase 1 clinical study describedbelow. Hence the full human dose as used in Phase 1 was administered inthe rat GLP toxicology study. The study was performed with a nonclinicalbatch which was representative for the batch used in the Phase 1clinical study.

No mortalities were observed during the study, nor any treatment relatedclinical signs (including body temperature) or ophthalmologicalobservations. Furthermore there were no toxicologically relevant,adverse effects on body weight, body weight gain, food consumption, orhematology, clinical chemistry, coagulation, and urinalysis parameters.There were no test article-related macroscopic findings or differencesin organ weights at the end of the treatment (Day 14) and the recoveryperiod (Day 28).

No adverse test article-related microscopic findings were observed.Non-adverse minimal to mild microscopic findings (interstitialinflammation, degeneration/necrosis of myofiber and mixed inflammatorycell infiltrates) were noted at the injection sites in the quadricepsfemoris muscle at the end of the treatment period in both the vehicleand treated group. These findings were therefore considered to be notrelated to administration of the test article, but a result of thedosing procedure (ie, i.m. injection). At the end of the recoveryperiod, Day 1-injection site muscles had recovered, while at the Day14-injection site, residual minimal mixed cellularinflammation/infiltrates were seen in the muscle, suggesting ongoingrecovery in both vehicle and treated animals. Overall, the vaccine waswell tolerated and no adverse treatment-related effects were noted.

Immunogenicity of the vaccine has been confirmed, inducing higher serumimmunoglobulin G (IgG) titers towards the 4 O-antigens in the vaccinatedgroup compared with vehicles that received only formulation buffer.

Repeated Dose Toxicity Study in Rabbits

A GLP repeated dose toxicity study with a 3-week recovery period wasconducted in NZW rabbits (TOX11163, draft report) to assess the toxicityand local tolerance of ExPEC4V following 3 i.m. injections (Days 0, 14,and 28) given 2 weeks apart. Reversibility, persistence, and delayedoccurrence of any changes were assessed on Day 49, after a 3-weektreatment-free period following the 3rd injection on Day 28. The designof the Phase 2-enabling repeated dose toxicity study in the rabbit canbe found in Table 5.

TABLE 5 Design of Repeated Dose Toxicity Study in the Rabbit Volume Doselevel^(a) Concentration injected per Dosing Number of Animals Group(μg/dose) (μg/mL) dosing ADM^(d) days Terminal^(e) Recovery^(e) 1 — 0 2× 1 mL ADM 1, 2 Day 0, 5 M + 5 F 5 M + 5 F ADM 3, 4 Day 14, ADM 5, 6 Day28 2 32^(b) 32    1 mL ADM 1 Day 0, 5 M + 5 F 5 M + 5 F ADM 3 Day 14,ADM 5 Day 28 3 64^(c) 32 2 × 1 mL ADM 1, 2 Day 0, 5 M + 5 F 5 M + 5 FADM 3, 4 Day 14, ADM 5, 6 Day 28 ADM: administration site; F = female; M= male Note: Day 0 = start of treatment; Group 1 received saline(control group) ^(a)Total O-antigen polysaccharide (1:1:1:1 ratio forO1A, O2, O6A and O25B serotypes, respectively) ^(b)8 μg polysaccharideper serotype + 109 μg total EPA carrier protein ^(c)16 μg polysaccharideper serotype + 218 μg total EPA carrier protein ^(d)ADM1: left - lowerpart m. biceps femoris; ADM2: right - lower part m. biceps femoris;ADM3: left - upper part m. biceps femoris; ADM4: right - upper part m.biceps femoris; ADM5: left - m. quadriceps femoris; ADM6: right - m.quadriceps femoris ^(e)Terminal animals were euthanized on Day 30 andrecovery animals on Day 49

A maximum dose of 16 μg per O-antigen PS of ExPEC4V (total PS dose of 64μg, together with 218 μg EPA carrier protein) was tested in this study.This dose is 4 times higher than the maximum dose that was testedpreviously in the Phase 1-enabling GLP toxicity study in the rat and isequivalent to the maximum PS (and EPA) dose that is evaluated in thePhase 2 clinical study described below. Hence the full (maximum) humandose as to be used in Phase 2 was administered in this rabbit GLPtoxicology study.

The study was performed with the ExPEC4V (containing 32 μg/mL total PS)that was used in the Phase 1 clinical study described below. This batchis considered representative for the vaccine composition that is used inthe Phase 2 clinical study described below (containing 128 μg/mL totalPS), as the same drug substances are used for both vaccine compositions.

No mortalities were observed during the study. There were no effects onbody temperature, body weight, body weight gain, food consumption,ophthalmology, skin evaluation (Draize scoring), clinical chemistry andC-reactive protein.

Females receiving 64 μg ExPEC4V exhibited non-adverse, minimallydecreased hemoglobin levels at the end of the treatment and recoveryperiod, with minimal decreases in total RBC count and hematocrit at theend of the recovery period. Fibrinogen was minimally increased 1 weekafter the 1st injection and at the end of the treatment period infemales, but no changes were observed anymore at the end of thetreatment-free (recovery) phase.

Shortly after dosing, dark discoloration of the subcutis was noted insome animals of the ExPEC4V-dosed groups, which correlated with thefoci/areas of discoloration that were seen at the sites injected on Day28 in all groups (including control group) at necropsy (Day 30). Noabnormalities were noted at the end of the recovery period.Histopathologically multiple foci of mixed inflammatory cell infiltrates(minimal to slight) were seen mainly at the Day 28 injection sites inExPEC4V-dosed animals. At the end of the recovery period (Day 49) only 1female in the high dose group exhibited mixed inflammatory cellinfiltrates at the injection sites of Day 28, indicating (ongoing)recovery.

Within the draining medial iliac lymph nodes, production (in germinalcenters) and sequestration of lymphoblastic cells was seen within theparacortex and/or medullary cords of ExPEC4V -dosed rabbits at the endof the treatment period, resulting in increased overall cellularity.Furthermore lymph nodes were larger in both sexes which correlated withan increased weight in females. These findings were not seen at the endof the recovery period. An increased number of germinal centers wasnoted in the spleen of treated males and females at the end of thetreatment and recovery period, and was accompanied by an increase inspleen weight in both sexes at the end of the treatment period.

These findings are considered non-adverse and related to the immuneresponse to the vaccine administration.

Immunogenicity of the vaccine was confirmed as serum IgG levels againstall 4 O-antigen serotypes as well as EPA were elevated in males andfemales.

Overall, vaccination of rabbits (3 i.m. injections, 2 weeks apart) withExPEC4V doses containing up to 64 μg total PS was safe and welltolerated. All treatment-related effects observed are considered toreflect a normal, non-adverse response induced by the vaccineadministration.

Functionality of Antibody Responses Induced by Vaccines in Rats

To assess the functional activity of vaccine-induced antibody responsesof O25B, O1A, O2 and O6A bioconjugates, sera from rats vaccinated withmonovalent or tetravalent vaccines containing O25B, O1A, O2 and O6A EPAbioconjugates, each alone or in combination, were analyzed usingopsonophagocytic killing (OPK) assays, which measure in vitrocomplement- and antibody-dependent phagocytosis and killing of bacteria,e.g., E. coli. The OPK assay measures the ability of serum to facilitateopsonophagocytosis and killing of different E. coli serotypes. In96-well plates, defined dilutions of the sample sera were incubated, ineach well, with bacteria from one of the four vaccine-specific E. coliserotypes, a defined amount of HL60 cells, and baby rabbit complement.After incubation, a proportion of the mixture was spotted onto trypticsoy agar (TSA) and the number of bacterial colonies was counted. Theability of the antibodies to bind the bacterial cells and activatedeposition of the complement and mediate uptake and killing of thebacteria by HL60 cells was expressed as opsonic titer. The opsonic titeror opsonization index (OI) corresponds to the dilution of the serakilling 50% of the bacterial cells. Opsonic indices for pre- andpost-immune sera are provided. At least a 4-fold increase of OI frompre- to post-immune is considered significant.

E. coli was pre-opsonized with dilutions of serum from vaccinated rats,incubated with complement and phagocytes (differentiated HL60 cells),and the colony forming units (CFUs) were determined. Subsequently, themaximum % killing and Opsonization Indices (OI: serum dilution killingof 50% of E. coli) were calculated. E. coli selected for OPK testingwere OC 24453 (serotype O2), OC 24781 (serotype O6A) and OC 24176(serotype O25B).

As shown by the results depicted in FIGS. 3A-3C, monovalent vaccinescontaining O2-EPA, O6A-EPA and O25B-EPA induced robust antibodyresponses in rats, and such antibody responses are functional in killingE. coli from these serotypes.

Table 6 shows the total OI titers for the O-antigens O2, O6A and O25Bfrom rats immunized with the tetravalent vaccine with either 0.4 or 4 μgper O-antigen. The titers were determined in two separate experiments.The 0.4 μg dose induced significant OIs in all animals for the O2 andO6A serotypes. For O25B, ⅜ animals showed a significant increase in OIfollowing immunization with the 0.4 μg dose. Compared to the 0.4 μgdose, the 4 μg dose induced lower OI increases for O2 in all animals. ⅜animals showed OI increases when the sera from the 4 μg dose group weretested on O25B E. coli.

The data confirm that a tetravalent vaccine is able to elicitO-antigen-specific opsonic antibodies against O2, O6A and O25B inanimals, demonstrating that the vaccine compositions described hereininduce antibody responses against E. coli serotypes from whichO-antigens are included in the vaccine, and that such antibody responsesare functional in killing E. coli from these serotypes.

TABLE 6 OIs against E. coli O2, O6A and O25B. OIs for individualpre-vaccination and post 3 vaccination sera from two separateexperiments are shown for all animals. Tetravalent-EPA Rat SerumOpsonization Indices (OI) O2 E. coli O6 E. coli 0.4 ug Dose 4 ug Dose0.4 ug Dose Animal No. Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 1:Pre-vacc 6 7 5 0 17  6 Postvacc >16384     1′476    293 32 202  226  2:Pre-vacc 21  11  11 20 11 90 Postvacc 11′148    >16384     150 120 436 475  3: Pre-vacc 6 6 0 0  0  5 Postvacc 11′073    >16384     46 19 98 374: Pre-vacc 5 5 5 6 23 17 Postvacc >16384    63  57 45 108  116  5:Pre-vacc 7 0 0 4 30  8 Postvacc 10′413    7′050    105 108 >16,384     12′672    6: Pre-vacc 8 0 8 7 299  164  Postvacc 89  34  24 17 1′725  1′475   7: Pre-vacc 9 9 6 6 18 21 Postvacc >16384     >16384     109 921′249   1′863   8: Pre-vacc 4 6 6 5 26 22 Postvacc 5′058    4′201    3925 6′590   3′826   Pre-vacc Av 8 5 5 6 53 42 Post-vacc Av 10′867   7′747    103 57 3′349   2′586   Tetravalent-EPA Rat Serum OpsonizationIndices (OI) O6 E. coli O25 E. coli 4 ug Dose 0.4 ug Dose 4 ug DoseAnimal No. Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 1: Pre-vacc 6 16 2′404    2′082    0 0 Postvacc 2′045    2′821    1′847    1′578    9 02: Pre-vacc 0 0 0 0 0 0 Postvacc 10′262    11′460    0 0 4 0 3: Pre-vacc0 0 0 0 0 0 Postvacc 7′959    8′597    6 0 355 197 4: Pre-vacc 0 0 0 0 00 Postvacc 2′189    4′488    0 0 70 26 5: Pre-vacc 8 7 0 0 0 0 Postvacc3′107    7′564    0 0 105 69 6: Pre-vacc 5 0 269  154  0 0 Postvacc 540 896  0 0 0 0 7: Pre-vacc 22  5 0 0 0 0 Postvacc 160  143  1′130    630 9 8 8: Pre-vacc 0 0 0 0 0 0 Postvacc 288  656  3′336    1′986    0 0Pre-vacc Av 5 3 334  280  0 0 Post-vacc Av 3′319    4′578    790  524 69 37

Effects in Human—Phase I Study

ExPEC4V has been tested in a first-in-human Phase 1 study, whichenrolled a total of 194 subjects. This Phase 1 study is a randomized,placebo-controlled, multicenter study. The study was conducted in asingle-blind manner as the investigator and study staff knew therandomization group of the subjects while the subjects were required tobe blinded to their randomization group at all times.

The objective of this first-in-human study was to evaluate the safety,immunogenicity, and efficacy of the candidate vaccine in healthy womenaged ≥18 to ≤70 years with a history of recurrent urinary tractinfection (rUTI). The primary objective of the study was the comparisonof solicited and unsolicited adverse events (AEs) and serious adverseevents (SAEs) between subjects who received ExPEC4V and subjects whoreceived placebo. The main secondary objectives included immunogenicityparameters, the number of symptomatic UTI episodes caused by E. colivaccine-serotypes, and the rate of occurrence and clinical symptoms ofvaccine-serotype-specific E. coli UTI.

Eligible subjects were required to have at least 3 independent UTIepisodes in the last 12 months or at least 2 independent UTI episodes inthe previous 6 months; at least 1 of the independent UTI episodes had tobe due to a culture-confirmed E. coli infection. A total of 194 subjectswere enrolled and randomized to a single i.m. dose of 0.5 mL of ExPEC4Vor placebo (see Table 7). The enrollment was done in a staggeredapproach to assess Day 14 safety data before proceeding to the nextphase:

1. The first 8 subjects were randomized (3:1) to a dose of 1 μg of eachPS, or placebo2. The following 8 subjects were randomized (3:1) to a dose of 4 μg ofeach PS, or placebo3. The remaining 178 subjects were randomized (1:1) to a dose of 4 μg ofeach PS, or placebo.

TABLE 7 the demographic and baseline characteristics of the subjectsenrolled in the Phase 1 study. ExPEC4V 1 μg polysaccharide 4 μgpolysaccharide Placebo per serotype^(a) per serotype^(a) Total N = 95 N= 6 N = 93 N = 194 Age (years) N 95 6 93 194 Mean (SD) 42.1 (15.9) 281(9.6) 42.0 (17.6) 41.6 (16.7) Median (range) 42.6 (18, 71) 23.1 (20, 43)38.5 (19, 72) 39.6 (18, 72) Race N 95 6 93 194 Caucasian 91 (96%) 6(100%) 87 (94%) 184 (95%) Other 4 (4%) 0 6 (6%) 10 (5%) Weight (kg) N 956 93 194 Mean (SD) 63.1 (11.0) 59.7 (10.3) 63.8 (11.0) 63.3 (10.9)Median (range) 61 (46.2, 95.0) 56.5 (50, 79) 63 (44, 105) 61 (44, 105)Body Mass Index (kg/m²) N 95 6 93 194 Mean (SD) 23.2 (4.1) 21.1 (4.3)23.3 (3.8) 23.2 (3.9) Median (range) 22 (17.8, 33.7) 19.9 (17.5, 29.4)22.7 (17.6, 34.3) 22.2 (17.5, 34.3) Menopausal Status N 95 6 93 194Premenopausal 64 (67%) 6 (100%) 57 (61%) 127 (65%) Child BearingPotential N 64 6 57 127 Yes 55 (86%) 6 (100%) 54 (95%) 115 (91%) ^(a)TheExPEC4V doses contain O-antigen polysaccharides of the 4 ExPEC serotypesO1A, O2, O6A, and O25B

At first visit, eligible subjects that have provided informed consentwere screened and compliance for inclusion/exclusion criteria wasconfirmed. Blood was drawn and urine was collected. At visit 2 (day 1),each subject received one intramuscular injection of 0.5 ml of solution(ExPEC4V or placebo) in the deltoid muscle. The reduced dose of thecandidate vaccine contained 1 μg of each polysaccharide (total 4 μgpolysaccharide). The target dose of the candidate vaccine contained 4 μgof each polysaccharide (total 16 μg polysaccharide).

Safety was evaluated based on solicited local (pain, erythema, andswelling at the injection site) and systemic (fever, i.e., bodytemperature ≥38° C.) AEs collected in a diary from Day 1 postvaccinationuntil Day 7 and on AEs and SAES collected until Day 270 (end of studyvisit). Immunogenicity was evaluated by qualified enzyme-linkedimmunosorbent assays (ELISA) and opsonophagocytic killing (OPK) assaysusing serum from blood samples taken prevaccination on Day 1 andpostvaccination on Days 30 and 270.

Descriptive statistics (n, mean, standard deviation, median and rangesfor continuous variables, frequencies and percentages for categoricalvariables) are provided by treatment group and/or visit, whereapplicable. All data are listed by subject, treatment group and, whereapplicable, visit. All subjects from Group B receiving placebo arecombined to form the placebo treatment group.

Safety

To date, none of the following has been identified from the Phase 1study (which is still on-going): adverse drug reactions, significantclinical laboratory abnormality, cardiovascular, pulmonary, centralnerve system, renal, or other significant adverse effects, overdose.Occurrence of adverse events and severe adverse events were comparablebetween the placebo and vaccinated groups.

Immunogenicity—Total Antibody Titer

To assess the immunogenicity of the vaccine components, sera from womenparticipating in the clinical study were obtained and analyzed by ELISAto quantify IgG against the four different O-antigens included in thetetravalent vaccine (E. coli O1, E. coli O2, E. coli O6, and E. coliO25B). Total Day 1 (prevaccination) and Day 30 serum IgG antibody titerswere assessed by a qualified ELISA optimized for each serotype isolateusing purified serotype-specific O-antigen as primary assay antigen.Total IgG antibody titers per serotype were calculated using a4-parameter logistic curve fit to determine per sample half maximaleffective concentration (EC₅₀) values.

1 μg Polysaccharide per Serotype in ExPEC4V (N=6)

Six subjects received an ExPEC4V dose of 1 μg PS per serotype (4 μgtotal PS). Analysis of serotype-specific immune responses of thesesubjects by Day 30 showed the proportion of subjects with a ≥2-foldincrease in total antibodies per serotype was 50% (serotype O1A), 83%(serotype O2), 50% (serotype O6A), and 67% (serotype O25B). Theproportion of subjects with a ≥4-fold increase in antibody titers waslower, i.e., 17% (serotype O6A), 33% (serotype 1A), 33% (serotype 2) and50% (serotype O25B).

Analysis of responses of the 1 μg PS per serotype dose group yieldedsimilar magnitude increases across the 4 serotypes. For serotypes O1A,O2, O6A, and O25B comparing Day 30 to Day 1, median fold increases inantibody titers were 2.5, 3.7, 2.2, and 4.1, respectively. Individualsubject fold increases ranged from 1 to 6 for serotypes O1A and O2, from1 to 7 for serotype O6A, and from 1 to 11 for serotype O25B. Geometricmean titer (GMT) values on Day 30 were 4,053, 13,768, 1,236, and 227 forthe respective 4 serotypes (Table 8), representing approximately a2.2-3.5 fold increase over Day 1 GMT values.

TABLE 8 GMT and 95% Confidence Intervals in ELISA-Determined TotalAntibody Titers from Day 1 (Prevaccination) to Day 30 - Phase 1 StudyExPEC4V 1 μg polysaccharide 4 μg polysaccharide Placebo per serotype^(a)per serotype^(a) N = 95 N = 6 N = 93 Antibody Day 1 Day 30 Day 1 Day 30Day 1 Day 30 O1A GMT 1,895 1,887 1,720 4,053 1,807 9,460 95% CI1,515-2,369 1,517-2,347  633-4,672 2,160-7,605  1,489-2,191 7,511-11,916 O2 GMT 3,529 3,502 4,266 13,768  2,855 27,973 95% CI2,926-4,257 2,910-4,214 1,731-10,511 6,516-29,091 2,279-3,57622,026-35,526 O6A GMT 943 953   558 1,236 920 4,475 95% CI   777-1,145  789-1,151  292-1,067  799-1,911   743-1,138 3,608-5,549 O25B GMT 285282   64   227 261 2,164 95% CI 211-384 209-381 16-254 53-976 188-3631,676-2,794

4 μg Polysaccharide per Serotype ExPEC4V (N=93)

In comparison to the dose containing 1 μg PS per serotype (4 μg totalPS), administration of an ExPEC4V dose of 4 μg PS per serotype (16 μgtotal PS) yielded a more robust immune response. Analysis of thesesubjects by Day 30 showed the proportion of subjects with a ≥2-foldincrease in total antibodies per serotype was 81% (serotype O1A), 92%(serotype O2), 80% (serotype O6A), and 82% (serotype O25B). Theproportion of subjects with a ≥4-fold increase in antibody titers rangedfrom 57% (serotypes O1A and O6A) to 80% (serotype O2), which is lowerthan the proportion with a 2-fold increase, but notably higher thanobserved with the dose containing 1 μg PS per serotype. See also FIG. 4, a robust immune response to each of O1A, O2, O6A, and O25B wasobserved, and that a significant increase in the ELISA titers betweenpost (30 days after injection) and pre-injection (day 1) was observedonly in the vaccinated groups (V_Day 30 v.s. V_Day 1), but not in theplacebo groups (P_Day 30 v.s. P_Day 1).

Analysis of responses of the 4 μg PS per serotype group yielded medianfold increases that were larger than those of the 1 μg PS per serotypedose group. For serotypes O1A, O2, O6A, and O25B comparing Day 30 to Day1, EC50 median fold increases were 4.6, 9.4, 4.9, and 5.9, respectively.The magnitude and variability in the fold increases were greater withthis dose group, ranging from 1 to 96 for serotype O1A, from 1 to 165for serotype O2, from 0 to 61 for serotype O6A, and from 1 to 579 forserotype O25B. GMT values on Day 30 were 9,460, 27,973, 4,475, and 2,164for the respective 4 serotypes (Table 8), representing approximately a4.9 to 9.8 fold increase over Day 1 GMT values.

CONCLUSION

These interim results show a vaccine-specific immune response in healthysubjects administered the 1 μg PS per serotype ExPEC4V dose, and acomparatively greater increase in the immune response with the higher 4μg PS per serotype ExPEC4V dose, over a 30-day observational period. Thelack of relevant change in antibody titers of the 95 subjects in theplacebo group over this period suggests the antibody response in ExPEC4Vrecipients is vaccine-mediated, and is not due to environmental exposureto ExPEC bacteria.

These data indicate an overall increase in the antibody titersassociated with the higher 4 μg PS per serotype dose compared to the 1μg PS per serotype dose. Although these results suggest greatervariability associated with the titers of the higher dose group comparedto the lower dose group, the small number of subjects in the 1 μg persubject dose limit the interpretation of any observed differences.Differences were also observed within each dose group in the relativeIgG titers per serotype, with the titer for antibodies to the O25Bantigen being the lowest (Table 8).

Functional Antibody Response

OPK assays were used to assess the functional antibody response of womenparticipating in the clinical study. Sera were collected from studyparticipants. E. coli was pre-opsonized with dilutions of serum from thevaccinated women, incubated with complement and phagocytes(differentiated HL60 cells), and the remaining colony forming units(CFUs) was determined. Subsequently, the maximum percent killing andOpsonization Indices (OI: serum dilution killing of 50% of E. coli) werecalculated. E. coli selected for OPK testing were OC 24452 (serotypeO1A), OC 24453 (serotype O2), OC 24454 (serotype O6A), and OC 24176(serotype O25B).

Day 1 and Day 30 sera were assessed for functional antibodies (measuredas the opsonization index [OI], or serum concentration yielding a 50%decrease in E. coli colony forming units) by an OPK assay, optimizedusing selected serotype O1A, O2, O6A, or O25B ExPEC strains, with humancomplement and HL60 phagocytic cells. Functional antibody titers forserotype O25B are included in the interim analysis based on preliminaryassessments of titer accuracy and reproducibility. For all serotypes,functional antibody titers are determined from measurements of E. coliopsonophagocytic-mediated killing using the NICE program, developed bythe U.S. National Institute of Standards and Technology, and theOpsititer3 program, developed and licensed from the University ofAlabama. For the interim analysis, OPK titers were determined for the194 subjects, including 95 subjects receiving placebo, 6 subjectsreceiving the ExPEC4V 1 μg PS (per serotype) vaccine and 93 subjectsreceiving the ExPEC4V 4 μg PS (per serotype) vaccine.

Placebo Recipients (N=95)

As observed with ELISA testing, placebo recipients (95 subjects) showedsimilar OPK responses for Day 1 and Day 30 sera vs ExPEC4V serotypes,with little or no observed change to most respective per-subject OPKtiter values. These results indicate a stable functional antibody titerfor most or all placebo subjects over this time period.

1 μg Polysaccharide per Serotype ExPEC4V (N=6)

Six subjects received an ExPEC4V dose of 1 μg PS per serotype (4 μgtotal PS). Analysis of serotype-specific immune responses of thesesubjects by Day 30 showed the proportion of subjects with a ≥2-foldincrease in total antibodies per serotype was 33% (serotype O1A), 67%(serotype O2), and 0% (serotypes O6A and O25B). For serotypes O1A andO2, the proportion of subjects with a ≥4-fold increase in antibodytiters decreased to 17% and 50%, respectively.

For serotypes O1A, O2, O6A, and O25B comparing Day 30 to Day 1, medianfold increases in antibody titers were 1.0, 4.8, 0.9, and 1.0,respectively. Individual subject fold increases ranged from 0.6 to 8.1for serotype O1, from 0.3 to 9.5 for O2, from 0.8 to 1.3 for serotypeO6A, and from 0.5 to 1.5 for serotype O25B. Geometric mean titer (GMT)values on Day 30 were 429, 1834, 1,136, and 51 for the respective 4serotypes, representing approximately a 1.0-2.8 fold increase over Day 1GMT values.

4 μg Polysaccharide per Serotype ExPEC4V (N=93)

Administration of an ExPEC4V dose of 4 μg PS per serotype (16 μg totalPS) yielded a functional immune response for all ExPEC 4V serotypes.Analysis of these subjects by Day 30 showed the proportion of subjectswith a ≥2-fold increase in OI values per serotype was 63% (serotypeO1A), 90% (serotype O2), 33% (serotype O6A), and 55% (serotype O25B).The proportion of subjects with a ≥4-fold increase in OI values rangedfrom 20% (serotype O6A) to 82% (serotype O2); as expected, theseproportions were consistently lower than those observed for the ≥2-foldincrease.

For serotypes O1A, O2, O6A, and O25B comparing Day 30 to Day 1, OImedian fold OPK titer increases were 3.5, 14.7, 1.4, and 2.5,respectively. The magnitude of the per subject fold increases with thisdose group ranged from 0.5 to >292 for serotypes O1A and O2, from 0.3 to26.4 for serotype O6A, and from 0.1 to 272.8 for serotype O25B. Asdepicted in FIGS. 5A-5D, a robust functional immune response to each ofO1A, O2, O6A, and O25B was observed, and a significant increase in theOI between post- and pre-injection was observed only in the vaccinatedgroups, not the placebo groups.

GMT values on Day 30 were 950.5, 4,132, 1,542, and 414.7 for therespective 4 serotypes (Table 9), representing approximately a 2- to14-fold increase over Day 1 GMT values. These data indicate an overallincrease in the Day 30 functional antibody titers associated with the 4μg PS per serotype dose, across all ExPEC 4V serotypes.

TABLE 9 GMT and 95% Confidence Intervals in OPK-Determined FunctionalAntibody Titers from Day 1 (Prevaccination) to Day 30 - Phase 1 StudyOpsonization Index^(a) ExPEC4V 1 μg polysaccharide 4 μg polysaccharidePlacebo per serotype^(b) per serotype^(b) N = 95 N = 6 N = 93 AntibodyDay 1 Day 30 Day 1 Day 30 Day 1 Day 30 O1A GMT 156 161 288 429 192.5950.5 95% CI 126.4-192.5   131-197.8 94-881 203-910 158.9-233.3  692.8-1,304.1 O2 GMT 341.9 341.6 652 1834 301.3 4,132 95% CI275.2-424.8 273.8-426.3 214-1980  425-7913 237.6-382.2 3,034.9-5,625. O6A GMT 692.6 715.1 1147 1136 790.6 1,542 95% CI 589.4-813.8 614.5-832.2794-1657  749-1721 676.7-923.7 1,263.7-1,881.  O25B GMT 86.6 97.3 52 51114 414.7 95% CI  58.4-128.5  64.3-147.2 27-103 28-92  74.6-174.5273.5-629  

CONCLUSION

These interim results show a vaccine-specific functional immune responsein healthy subjects administered the 4 μg PS per serotype ExPEC4V dose,over a 30-day observational period. The lack of significant change inantibody titers of the 95 subjects in the placebo group over this period(FIGS. 5A-5D and Table 9) is consistent with ELISA results and supportsthe conclusion that an antibody response to vaccination has beendemonstrated across the 4 ExPEC serotypes.

Interim immunogenicity results from the Phase 1 study show for bothtotal (ELISA) and functional (OPK) antibody titers a vaccine-specificimmune response in healthy subjects administered the 1 μg PS perserotype ExPEC4V dose, and a comparatively greater increase in theimmune response with the higher 4 μg PS per serotype ExPEC4V dose, overa 30-day observational period. Comparison with serotype-specific foldincreases observed with ELISA testing, the OPK assay showed similarresponse levels for serotypes O1A and O2 for both assays, but somewhatlower levels of OPK % subject responses with serotypes O6A and O25B. Theselective decrease in OPK response of some subjects for serotypes O6Aand O25B is under investigation.

Notably, the GMT value in OPK-determined functional antibody titer forO25B antigen is lower than those for the other antigens (O1A, O2 andO6A). The OPK assay has been accepted as a better surrogate assay forimmune protection induced by the PS conjugate vaccine againstStreptococcus pneumoniae (Prevenar®), since the ELISA may notdifferentiate nonprotective low-avidity antibodies from protectivehigh-avidity antibodies (Kim et al., Clin Diagn Lab Immunol. 200310(4):616-21)

Effects in Human—Phase II Study

Based on the interim results from the Phase I study, a Phase II studywill be conducted. This randomized, double-blind, placebo-controlledmulticenter study is planned to evaluate safety, tolerability, andimmunogenicity of 5 different doses in men and women in stable health,stratified by age: ≥18 to <50 years old (N=275) and ≥50 years old(N=560).

Two vaccine compositions, i.e., Products 1 &2 provided in Table 3-2,having two different polysaccharide concentrations using the same activesubstances as those used in ExPEC4V will be used in the Phase II study.More specifically, the Composition 1 formulation contains 32, 32, 32, 32μg/ml per O-antigen polysaccharide (PS) of the E. coli serotypes O1A,O2, O6A and O25B, respectively, without adjuvant. The Composition 2formulation contains 16, 16, 16, 32 μg/ml per O-antigen PS of the E.coli serotypes O1A, O2, O6A and O25B, respectively, without adjuvant.

Different dosages and ratios of the active substances will be tested inthe phase II study. More specifically, the enrolled subjects will berandomized and divided into six arms: (i-v) five different doses ofcandidate vaccine and (vi) placebo. Each subject will receive a singlei.m. dose of Composition 1, Composition 2 or placebo. The target dosefor the E. coli O-antigens O1A: O2: O6A: O25B per injection ofComposition 1 or 2 is: 4:4:4:4 μg (i.e., the same as the highest doseused in the phase I study above), 4:4:4:8, 8:8:8:8, 8:8:8:16 and16:16:16:16 μg. The objective of the study is to assess the safety,immunogenicity, and efficacy of the 5 different doses of the tetravalentE. coli bioconjugate vaccine.

Sequences

SEQ Description SEQUENCE ID NO. Detoxified EPAGSGGGDQNATGSGGGKLAEEAFDLWN 1 protein ECAKACVLDLKDGVRSSRMSVDPAIAcomprising DTNGQGVLHYSMVLEGGNDALKLAID 4 optimized N-NALSITSDGLTIRLEGGVEPNKPVRY glycosylation SYTRQARGSWSLNWLVPIGHEKPSNIsequences KVFIHELNAGNQLSHMSPIYTIEMGD ELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWA SGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKDNN NSTPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCGY PVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALT LAAAESERFVRQGTGNDEAGAASADVVSLTCPVAKDQNRTKGECAGPADSGD ALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGY HGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARG RIRNGALLRVYVPRWSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAIT GPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISA LPDYASQPGKPPREDLKLGSGGGDQN AT N-glycosylationAsn-X-Ser(Thr), wherein X 2 consensus  can be any amino acid sequenceexcept Pro N-glycosylation Asp(Glu)-X-Asn-Z-Ser(Thr), 3 consensuswherein X and Z are sequence independently selected from any natural amino  acid except Pro

The embodiments described herein are intended to be merely exemplary,and those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. All such equivalents areconsidered to be within the scope of the present invention and arecovered by the following claims.

All references (including patent applications, patents, andpublications) cited herein are incorporated herein by reference in theirentirety and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

REFERENCES

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(3) Foxman, Am J Med. 2002; 113 Suppl 1A:5S-13S;

(4) Russo et al., Microbes Infect. 2003; 5(5):449-456

(5) Schito et al., 2009, Int. J Antimicrob. Agents 34(5):407-413

(6) Pitout et al., 2012, Expert Rev. Anti. Infect. Ther.10(10):1165-1176

(7) Johnson et al., Antimicrob Agents Chemother. 2010; 54(1):546-550

(8) Rogers et al., J Antimicrob Chemother. 2011; 66(1):1-14

(9) Banerjee et al., Antimicrob Agents Chemother. 2014; 58(9):4997-5004

(10) Stenutz et al., FEMS Microbial Rev. 2006; 30: 382-403

(11) Russo et al., Vaccine. 2007; 25: 3859-3870

(12) Lipsitch, Emerging Infectious Diseases; 1999, 5:336-345

(13) WO 2006/119987

(14) WO 2009/104074

(15) International Patent Application No. PCT/EP2015/053739 (publishedas WO 2015/124769)

(16) Ihssen et al., 2010, Microbial Cell Factories 9, 61

(17) Lukac et al., Infect Immun, 56: 3095-3098, 1988

(18) Ho et al., Hum Vaccin, 2:89-98, 2006

(19) Pawlowski et al., 2000, Vaccine 18:1873-1885

(20) Robbins et al., 2009, Proc Natl Acad Sci USA 106:7974-7978

(21) Saraswat et al., 2013, Biomed. Res. Int. ID #312709 (p. 1-18)

(22) WO/2009/104074

(23) Datsenko and Wanner (2000) Proc Natl Acad Sci USA 97: 6640-6645

(24) WO 2014/057109

(25) Laurentin and Edwards, 2003, Anal Biochem 315, 143-145

(26) Kim et al., Clin Diagn Lab Immunol. 2003 10(4): 616-21

1-20. (canceled)
 21. A composition comprising an E. coli O25B antigenpolysaccharide at a first concentration, and E. coli O1A, O2 and O6Aantigen polysaccharides each at a concentration that is independently40% or 50% of the first concentration, each of the E. coli O25B, O1A, O2and O6A antigen polysaccharides are independently covalently bound to acarrier protein, and the first concentration is 8 to 48 μg/ml.
 22. Thecomposition of claim 21, wherein each of the E. coli O1A, O2 and O6Aantigen polysaccharides have the same concentration.
 23. The compositionof claim 21, wherein each of the E. coli O1A, O2 and O6A antigenpolysaccharides have different concentrations.
 24. The composition ofclaim 21, comprising 16 μg/ml of the O25B antigen polysaccharide. 25.The composition of claim 21, comprising 32 μg/ml of the O25B antigenpolysaccharide.
 26. The composition of claim 21, wherein the firstconcentration is 10 to 36 μg/ml.
 27. A multivalent immune compositioncomprising an E. coli O25B antigen polysaccharide at a first dose of 5to 18 μg, and an E. coli O1A antigen polysaccharide, an E. coli O2antigen polysaccharide and an E. coli O6A antigen polysaccharide each ata dose that is independently 40% or 50% of the first dose, wherein eachof the E. coli O25B, O1A, O2 and O6A antigen polysaccharides areindependently covalently bound to a carrier protein.
 28. The multivalentimmune composition of claim 27, wherein the E. coli O25B antigenpolysaccharide comprises the structure of Formula O25B′:

the E. coli O1A antigen polysaccharide comprises the structure ofFormula O1A′:

the E. coli O2 antigen polysaccharide comprises the structure of FormulaO2′:

and the E. coli O6A antigen polysaccharide comprises the structure ofFormula O6A′:

wherein n is independently an integer of 5 to 25, and each of the E.coli O25B, O1A, O2 and O6A antigen polysaccharides are independentlycovalently bound to a carrier protein.
 29. The multivalent immunecomposition of claim 28, wherein the first dose is 16 μg.
 30. A methodof inducing an immune response to extra-intestinal pathogenic E. coli(ExPEC) in a subject, comprising administering to the subject acomposition of claim
 21. 31. The method of claim 30, wherein the subjectis a human.
 32. A method of inducing an immune response toextra-intestinal pathogenic E. coli (ExPEC) in a subject, comprisingadministering to the subject a first effective amount of an E. coli O25Bantigen polysaccharide, and a second effective amount of each of an E.coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharideand an E. coli O6A antigen polysaccharide, each of the E. coli O1A, O2and O6A antigens is administered at an effective amount that isindependently 40% or 50% of the first effective amount of the E. coliO25B antigen, each of the E. coli O25B, O1A, O2 and O6A antigenpolysaccharides are independently covalently bound to a carrier protein,and the first effective amount is 5 to 18 μg per administration, whereinsaid subject is a human.
 33. The method of claim 32, wherein 8 μg of theO25B antigen polysaccharide is administered per administration.
 34. Themethod of claim 32, wherein 16 μg of the O25B antigen polysaccharide isadministered per administration.
 35. The method of claim 32, wherein theE. coli O25B, O1A, O2 and O6A antigen polysaccharides are administeredtogether in one composition.
 36. The method of claim 32, wherein the E.coli O25B antigen polysaccharide comprises the structure of FormulaO25B′:

the E. coli O1A antigen polysaccharide comprises the structure ofFormula O1A′:

the E. coli O2 antigen polysaccharide comprises the structure of FormulaO2′:

and the E. coli O6A antigen polysaccharide comprises the structure ofFormula O6A′:

wherein n is independently an integer of 5 to 25, and each of the E.coli O25B, O1A, O2 and O6A antigen polysaccharides are independentlycovalently bound to a carrier protein.
 37. The method of claim 32,wherein the immune response limits the severity of or prevents aninvasive ExPEC disease caused by ExPEC serotypes O1A, O2 and O6A andO25B in an at-risk human subject.
 38. The method of claim 37, whereinthe at-risk human subject has or is at risk of having an invasive ExPECdisease selected from the group consisting of urinary tract infection, asurgical-site infection, an abdominal or pelvic infection, pneumonia,nosocomial pneumonia, osteomyelitis, cellulitis, sepsis, bacteremia, awound infection, pyelonephritis, meningitis, neonatal meningitis,peritonitis, cholangitis, soft-tissue infections, pyomyositis and septicarthritis.
 39. The method of claim 27, wherein each of the E. coli O1A,O2 and O6A antigens is administered at an effective amount that is 50%of the first effective amount of the E. coli O25B antigen.
 40. A processof making a composition of claim 32, comprising combining the E. coliO25B antigen polysaccharide, the E. coli O1A antigen polysaccharide, theE. coli O2 antigen polysaccharide and the E. coli O6A antigenpolysaccharide to thereby obtain the composition.